Gene Expression
Nebulas
Gene Expression NebulasSummary: Myelin-reactive T cells have been identified in patients with multiple sclerosis (MS) and healthy subjects with comparable frequencies, but the contribution of these autoreactive T cells to disease pathology remains unknown. A total of 13,324 T cell libraries generated from blood of 23 patients and 22 healthy controls were interrogated for reactivity to myelin antigens. Libraries derived from CCR6+ myelin-reactive T cells from patients with MS exhibited significantly enhanced production of IFN-b, IL-17, and GM-CSF compared to healthy controls. Single-cell clones isolated by MHC/peptide tetramers from CCR6+ T cell libraries also secreted more pro-inflammatory cytokines while clones isolated from controls secreted more IL-10. The transcriptomes of myelin-specific CCR6+ T cells from patients with MS were distinct from those derived from healthy controls, and of note, were enriched in Th17-induced experimental autoimmune encephalitis (EAE) gene signatures and gene signatures derived from Th17 cells isolated other human autoimmune diseases. These data, although not casual, imply that functional differences between antigen specific T cells from MS and healthy controls is fundamental to disease development and support the notion that IL-10 production from myelin-reactive T cells may act to limit disease progression, or even pathogenesis.
Overall Design: Four conditions of purified T cells with between 3 and 5 replicates per condition.
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| Growth Protocol: | CCR6+ memory CD4+ T cells from HLA-DR4+ healthy controls and HLA-DR4+ MS patients were amplified by PHA and IL-2 and stimulated by irradiated autologous monocytes and DR4 myelin peptides (MOG97-109 and PLP180-199). Cell proliferation was measured and two of the highest proliferated wells were picked for DR4 tetramers staining (MOG97-109-tetramers and PLP180-199-tetramers). Myelin tetramer+ and tetramer− cells were sorted into RNA lysis buffer for RNA sequencing. |
| Treatment Protocol: | Amplified T cell libraries assay were carried out as previous described (Geiger, 2009). Naïve, CCR6− and CCR6+ memory CD4+ T from paired MS patients and healthy controls were pre-sorted and cultured in 96-well round-bottom plates (Costar) at 2 × 103 cells per well in complete RPMI 1640 medium, and stimulated with 1 μg/ml PHA (Roche) and 20 U/ml IL-2 in the presence of irradiated (45Gy) allogeneic feeder cells (2×104/well). IL-2 was added on day 4, 7 and 10. Cultures were washed and split into two 96-well plates after 2-weeks of stimulation and expansion. Library screening was performed by culturing ~ 106 T cells/well with autologous monocytes (~105), which were either unpulsed or pulsed for 3 h with 10 μg/ml myelin peptide pools (MBP85-99, MOG222-241, PLP30-49, PLP129-148, MOG97-109, PLP180-199), or C. albicans (GREER). [3H] (Perkin Elmer) was added into the cultures 16 h before harvest. On day 5, cell proliferation was measured by 3H-thymidine incorporation on a scintillation beta-counter (Perkin Elmer). Culture supernatants were taken on day 7 for cytokine profiling as described below. |
| Extract Protocol: | RNA was extracted using the NucleoSpin RNA XS Kit (Macherey-Nagel, Bethlehem, PA) according to the manufacturers instructions. |
| Library Construction Protocol: | cDNA synthesis and amplification were performed using SMARTer Ultra Low Input RNA for Illumina Sequencing High Volume Kit (Clontech, Mountain View, CA) according to the manufacturers instructions. The average number of cells used to isolate RNA was 3,000, which yielded >1ng input RNA. Paired-end sequencing libraries were prepared using the Nextera XT DNA sample Prep Kit (Illumina, SanDiego, CA) according to the manufacturers instructions. |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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