Project description:Splenocytes were separated from Lrrc8af/f and Lrrc8af/f Cd4-Cre;OT-I mice. The splenocytes were stimulated with indicated peptides (unstime/ SIINFEKL-10pM(NL)/ SIINFEKL-1000pM(NH)/ SIIGFEKL-1000nM(G4)) for 6 hours. The OT-I CD8+ T cells were labeled with Va2-PE/CD8-A700 and sorted for the sequencing.
Project description:Activation of CD8+ T cells depends exquisitely on the affinity of the T cell receptor (TCR) for a peptide MHC (pMHC) ligand complex. Here, we activated OT-I transgenic CD8+ T cells with pure peptide and examined early activation responses by single-cell RNA-sequencing. T cells were activated with the high affinity OT-I cognate peptide (N4=SIINFEKL) for 1, 3 or 6 hours, or with reduced affinity peptides (T4=SIITFEKL and G4=SIIGFEKL) or the non-binding peptide (NP68=ASNENMDAM) for 6 hours. Cells were then sorted into 96-well plates by FACS and RNA was sequenced following an adapted Smart-Seq2 protocol.
Project description:Interleukin 6 (IL-6) is a pleiotropic cytokine with diverse roles in homeostasis, inflammation, and cancer. To identify transcriptional patterns and differentiation states induced by IL-6 in CD8 T cells, we performed RNAseq profiling of murine OT-I CD8+ T cells stimulated with SIINFEKL peptide in the presence of exogenous IL-6 (10 ng/ml) or anti-IL6R antibody (to block endogenous IL-6 signaling). Bulk OT-I splenocytes were stimulated with SIINFEKL peptide and CD8 T cells were FACS-sorted for RNA extraction after 2 and 7 days. We found that IL-6 potently repressed classical effector differentiation and promoted a gene expression pattern similar to that of memory precursor cells.
Project description:This experiment aims to study the transcriptome of activated T cells at 24h, using the same biological samples as the polysome profiling in GSE84781. A T cell receptor (TCR) transgenic mouse model (OT-1) posessing a homogenous alpha-beta CD8 T lymphocyte population was used, and cells with intact TCR signalling pathways (WT) were compared to those with defective signal transduction (Lck-/-, OFF). Ex vivo OT-1 T cells were isolated from the superficial cervicals, axillary, brachial, mesenteric, and inguinal lymph nodes and then activated for 24h with cognate peptide antigen SIINFEKL. Total RNA was extracted from the cells, labelled with Cy3, and hybridised onto an Agilent-074809 SurePrint G3 Mouse GE v2 8x60K Microarray.
Project description:In order to identify gene targets of translational regulation during T cell activation, a polysome analysis was performed in a T cell receptor (TCR) transgenic model (OT-1) possessing a homogenous alpha-beta CD8 T lymphocyte population. Cells with defective TCR signal tranduction (Lck-/-, OFF) were compared to WT cells to visualise targets regulated by TCR signalling. Total cytoplasmic ribonucleoprotein was extracted from ex vivo OT-1 transgenic T lymphocytes stimulated with SIINFEKL peptide antigen for 24h, extracts were fractionated using a sucrose density gradient, and separated into a sub-polysomal and a polysomal fraction. Total RNA from each fraction was extracted, equal volume of RNA from each fraction was individually labelled (Sub-poly: Cy3; Poly: Cy5), and hybridised onto a Agilent SurePrint G3 Mouse GE v2 8x60K Microarray.
Project description:OT-1 T-cells were co-cultured either alone in T-cell media supplemented with IL-2 (50IU/mL IL-2), with OVA loaded macrophages, or with both OVA loaded macrophages and CT2A-TRP2-β2mKO tumor cells. Cells were cultured at a 5:1 T-cell to tumor ratio, and 2:1 T-cell to macrophage ratio. After 24 h of co-culture, CD8 T-cells were FACS sorted, and RNA extracted (RNeasy Mini Kit, Qiagen). RNA was analyzed on an nCounter MAX Analysis System (Nanostring) with the PanCancer Immune profiling panel (Nanostring) according to manufacturer instructions. Expression dat OT-1 T-cells were isolated from OT-1 mice by culturing OT-1 splenocytes in T-cell media supplemented with 50IU/mL IL-2 and 1 μM OVA SIINFEKL peptide (Anaspec) for 48 h. Cells were purified for CD8 T-cells as described above and subsequently cultured in TCM with 50IU/mL IL-2, splitting every 24h for a total of 4 days.
Project description:The goal was to determine how IL-12 affects gene expression by murine CTL. Experiment Overall Design: Following RBC lysis, splenocytes from OT-1 TCR transgenic mice (5 million/ml) were cultured in six well-plates (5-6ml/well) in IMDM supplemented with 10% FCS and 55μM 2-ME with 1μM SIINFEKL. After 4 days, the cells were harvested by purification with Ficoll-Paque PLUS and placed in fresh medium containing 0 or 20ng/ml rmIL-12. Twenty-four hours later the cells were harvested and used in experiments. The live cells harvested were >98% CD8+Vα2+ (OT-1).
Project description:OT-I CD8 T cells primed with cognate SIINFEKL peptide and agonists to the costimulatory receptors CD134 and CD137 were stimulated with and without IL-2, and then STAT5 ChIP-seq was performed.
Project description:We performed RNA-Seq and compared expression levels of genes of NKG2D+ OT-II Th1 or Th17 cells to OT-II x NKG2D-/- Th1 and Th17 cells, respectively.
Project description:Single cell RNA-seq analyiss of FACS enriched OT-I CD8+ T cells following co-culture with SIINFEKL-pulsed bone marrow-derived DCs and bone marrow-derived myeloid-derived suppressor cells (T cell:DC:MDSC = 1:0.2:2)
