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SRP049335
Mus musculus
22 Downloadable Samples
Illumina HiSeq 2500
Description
Cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and Programmed death-1 (PD-1) are immunoregulatory receptors expressed on T cells that play important roles in suppressing immune responses to cancer. Although monoclonal antibodies that target CTLA-4 or PD-1 stimulate therapeutic anti-tumour T cell responses, the tumour antigens recognized by checkpoint blockade immunotherapy remain undefined. Herein, we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T cell rejection antigens following aPD-1 and/or aCTLA-4 treatment of mice bearing progressively growing sarcomas. We validate this conclusion by showing that (a) the predicted mutant epitopes associate with MHC class I molecules of the tumour; (b) T cells specific for these mutant epitopes infiltrate tumours; and (c) therapeutic vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Whereas, T cells with the same antigen specificity are present in progressively growing tumours in control mice, tumour-specific T cells in aPD-1- and/or aCTLA-4-treated mice express some overlapping but mostly treatment-specific transcriptional profiles that render them capable of tumour rejection. Thus, tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy but also can be used to identify tumour antigen-specific T cells that function as biomarkers of successful anti-tumour responses. Overall design: For sorting of mLama4-specific cells, tumour-infiltrating cells were enriched for CD45+ cells using CD45 cell purification magnetic beads (Miltenyi Biotec). CD45 enriched cells were then sorted gating for PI- CD3e+ CD8a+ mLama4-tetramer-PE+ or PI- CD3e+ CD8a+ mLama4-tetramer-PE- cells. Sorting was performed on a BD FACSAria II (BD Biosciences). Sorted cells were pelleted and processed for RNA analysis. All flow cytometry was performed on the FACSCalibur (BD Biosciences) or LSR Fortessa (BD Biosciences) and analysed using FlowJo software (TreeStar).
Publication Title
Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 27 Downloadable Samples
- Illumina HiSeq 2000
Description
In prior work we developed an optogenetic system for delivering highly precise, time-varying inputs to Ras, termed OptoSOS (Toettcher et al., 2013). This system relies on a membrane-targeted photoswitchable protein (Phy-CAAX) and a cytoplasmic Ras activator (PIF-SOScat) whose localization to the membrane can be controlled with light. In this system, Phy/PIF heterodimerization can be triggered on and off by exposure to 650 and 750 nm light, respectively. We found that this system could be used to deliver highly precise levels and dynamics of Ras/Erk signaling both in vitro and in vivo. Here, we aimed to globally assess the transcriptional response to light-activated Ras and compare it to that induced by growth factor stimulation. We stimulated NIH3T3 OptoSOS cells with either constant activating red light or PDGF and measured transcriptional responses by RNAseq. Total mRNA was collected after 0, 30, 60 and 120 minutes and used to track the dynamics of transcript abundance in both conditions. Genes were defined as upregulated if they satisfied two criteria: (i) induced at least three-fold over unstimulated cells, and (ii) induced at least two consecutive timepoints. By these criteria we detected 118 genes that were upregulated within 2 h by either PDGF or light stimulation, a comparable number of Ras-responsive genes to that found in previous studies. We found that both PDGF and light induced nearly identical profiles of gene expression, with 100/118 genes induced by PDGF and 110/118 induced by light. At each time point we found excellent agreement between the levels of gene induction in response to both stimuli. This agreement also extended to response dynamics. where hierarchical clustering revealed three classes of dynamic response: an early response peaking within 30 min, an intermediate response peaking at ~1 h, and a late response where gene expression gradually increased over the full 2 h timecourse. In all three classes, we found that light and PDGF led to highly similar expression changes over time. We thus concluded that sole stimulation of the Ras/Erk pathway by light was sufficient to recapitulate at least the first two hours of the PDGF-induced transcriptional response. Overall design: RNA-seq to measure global transcript abundance at various timepoints after PDGF stimulation or direct optogenetic activation of Ras using the OptoSOS system in NIH3T3 cells (Toettcher et al, Cell 2013). 9 samples were collected using the TruSeq library preparation kit (Illumina), multiplexed, pooled and measured in 3 lanes of an Illumina Hi-Seq 2000. Library quality was assessed by Agilent Bioanalyzer. Roughly 30-50 million reads were measured per sample across all 3 lanes. Baseline transcript abundance was measured in triplicate (0 min controls) and each successive timepoint was measured in a single collection. Genes were considered upregulated if they were induced at least 5-fold in at least two consecutive timepoints relative to their baseline abundance.
Publication Title
Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 113 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
A gene expression profiling sub-study was conducted in which colonic biopsy samples were collected for RNA extraction and hybridization to microarrays from 48 patients with UC who were participating in ACT 1, a placebo-controlled study of infliximab. Gene expression profiles from infliximab responders were compared with those of baseline and infliximab non-responder samples.
Publication Title
Gene expression profiling and response signatures associated with differential responses to infliximab treatment in ulcerative colitis.
Sample Metadata Fields
Subject, Time
View Samples- Rattus norvegicus
- 6 Downloadable Samples
- Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
Experimental autoimmune uveitis (EAU) in Lewis rats is a model for the clinical heterogeneity of human uveitis. The autoantigens inducing disease in the rat are also seen in human disease. Depending upon the specific autoantigen used, the experimental disease course can be either monophasic or relapsing/remitting and appears to be dictated by the T cell effector phenotype elicited. We investigated potential differences between monophasic and relapsing/remitting effector T cells using transcriptomic profiling and pathway analysis. RNA samples isolated from three independent T cell lines derived from each specificity where analyzed by microarrays.
Publication Title
Effector T cells driving monophasic vs. relapsing/remitting experimental autoimmune uveitis show unique pathway signatures.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 12 Downloadable Samples
- Affymetrix Human Genome U133A Array (hgu133a)
Description
Both spotted long oligonucleotide arrays (GPL1384) and Affymetrix GeneChip arrays (GPL96) were used to analyze gene expression in six human head and neck squamous cell carinoma samples versus control samples or lymph node metastases of the same patients. Hybridizations of HG-U133A GeneChip arrays were performed using standard Affymetrix protocols and equipment. Before hybridization on DKFZ Operon 27k long oligonucleotide arrays, 2 g RNA were amplified by one round of linear isothermal RNA amplification, followed by Cy-dUTP incorporation using Klenow fragment. Hybridizations were performed for 16 h at 42 C in a GeneTAC Hybridization Station (Genomic Solutions) using UltraHyb hybridization buffer (Ambion). Hybridized microarrays were scanned at 5 m resolution on a GenePix 4000B microarray scanner (Axon Instruments). Raw signal intensities from both platforms were normalized applying variance stabilization (W. Huber et al., Bioinformatics 18 Suppl 1, 2002). Expression ratios were compared for those genes represented in both array platforms.
Publication Title
Patient-based cross-platform comparison of oligonucleotide microarray expression profiles.
Sample Metadata Fields
No sample metadata fields
View Samples- Rattus norvegicus
- 17 Downloadable Samples
- Affymetrix Rat Genome U34 Array (rgu34a)
Description
Most kidney allograft losses are caused by chronic allograft dysfunction (CAD) that is characterized by interstitial fibrosis, tubular atrophy and a smoldering inflammatory process. The aim of the study was to correlate changes in gene expression over time, as evidenced by effects on regulatory pathways linked to the development of fibrosis and inflammation during the development of chronic damage. Renal allografts were harvested for time points 0d, 7d, 14d and 56d (n=3-5) and examined for steady state mRNA expression using Affymetrix microarray RG-U34A. A select group of genes previously associated with chronic fibrosis was then examined in the context of progressive dysfunction. In order to verify the microarray analysis, qPCR has been performed.
Publication Title
Wnt pathway regulation in chronic renal allograft damage.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 8 Downloadable Samples
- Affymetrix Murine Genome U74A Version 2 Array (mgu74av2)
Description
Selection of B cells subjected to hypermutation in germinal centres (GC) during T-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and T-independent germinal centre B cells. We have now compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC.
Publication Title
Axon growth and guidance genes identify T-dependent germinal centre B cells.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 30 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Infliximab, an anti-TNF-alpha monoclonal antibody, is an effective treatment for ulcerative colitis (UC) with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-TNF-alpha is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in UC.
Publication Title
Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis.
Sample Metadata Fields
Specimen part, Disease
View Samples- Homo sapiens
- 23 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Infliximab, an anti-TNFa monoclonal antibody, is an effective treatment for ulcerative colitis (UC) inducing over 60% of patients to respond to treatment. Consequently, about 40% of patients do not respond. This study analyzed mucosal gene expression from patients enrolled in ACT1 to provide a predictive response signature for infliximab treatment.
Publication Title
Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 16 Downloadable Samples
- Illumina HiSeq 1500
Description
Lymphocytic Choriomeningitis Virus (LCMV) specific CD8+ T cells (P14) were transferred into congenic WT mice followed by LCMV(DOCILE) infection. CXCR5-expressing (CXCR5+) or CXCR5 non-expressing (CXCR5-) P14 were purified on day 8 after infection, and total mRNA were sequenced from these populations. mRNA of P14 from uninfected mice (Naive P14) was also sequenced. Overall design: Examination of mRNA level in CXCR5 expressing P14 (CXCR5+P14) and non-expressing P14 (CXCR5-P14) from LCMV infected mice day 8 post infection. mRNA of P14 from uninfected mice (Naïve P14) was also examined.
Publication Title
CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.
Sample Metadata Fields
Subject
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