Project description:Inflammatory cytokines promote the accumulation of activated CD8 T cells. Here, we transfer 600 OT-I CD8 T cells iv into naïve C57BL/6 hosts. One day later, 500,000 LPS-matured and OVA257 peptide-coated DC were injected iv into OT-I CD8 T cell seeded hosts with (DC+CpG) or without (DC). Other seeded mice were infected with 2x10^4 virulent Listeria monocytogenes (vLM-OVA) iv. OT-I CD8 T cells were harvested from the spleen, flow sort purified, then RNA was extracted using RNeasy (Qiagen) kit. Naive OT-I CD8 T cells (Naive) were purified from the spleens of OT-I transgenic mice. Each group had three independent biological replicates.Transcriptomes were compared using DAVID analysis (with genes scoring FDR<0.01) and GSEA analysis. 3 biological replicates per group. Groups included Naïve OT-I CD8 T cells, DC+CpG OT-I CD8 T cells, DC OT-I CD8 T cells, and vLM-OVA OT-I CD8 T cells. Most comparisons used Naïve OT-I CD8 T cells as a baseline comparison
Project description:Inflammatory cytokines promote the accumulation of activated CD8 T cells. Here, we transfer 600 OT-I CD8 T cells iv into naïve C57BL/6 hosts. One day later, 500,000 LPS-matured and OVA257 peptide-coated DC were injected iv into OT-I CD8 T cell seeded hosts with (DC+CpG) or without (DC). Other seeded mice were infected with 2x10^4 virulent Listeria monocytogenes (vLM-OVA) iv. OT-I CD8 T cells were harvested from the spleen, flow sort purified, then RNA was extracted using RNeasy (Qiagen) kit. Naive OT-I CD8 T cells (Naive) were purified from the spleens of OT-I transgenic mice. Each group had three independent biological replicates.Transcriptomes were compared using DAVID analysis (with genes scoring FDR<0.01) and GSEA analysis.
Project description:To investigate the impact of TGFb on CD8 T cell activation and gene expression, we activated TCR transgenic OT-I T cells in the presence or absence of recombinant TGFb in vitro
Project description:We report the gene expression differences between OT-1 T cells activated alone or in the presence of TLR1/2 (Pam3CSK4) or TLR7 (Gardiquimod). By activating OT-1 splenocytes in the presence or absence of the TLR agonists, isolating RNA from purified CD8+ T cells we show that cells activated in the presence of either TLR1/2 or TLR7 agonists had similar transcriptional profiles demonstrating an increase in Th1 cytokine expression over cells that were activated alone. This study provides a key piece of information for those designing T cell mediated therapies.
Project description:NaM-CM-/ve, liver- and gut-activated CD8 OT-I T cells show differential migration behaviour. To analyze which genes could be responsible for different migration patterns, naM-CM-/ve, liver-activated and gut-activated CD8 T cells were isolated and compared for their gene expression profile. Gene expression profiles of naM-CM-/ve CD8 OT-I T cells versus liver-activated T cells and gut-activated T cells, respectively. NaM-CM-/ve CD8 OT-I T cells were isolated from lymph nodes and spleens of OT-I mice. CD8 OT-I T cells activated by liver-derived and gut-derived antigen for three days in vivo, positive for CFSE were sorted from livers of TF-OVA mice or from mesenteric lymph nodes of iFABP-OVA mice. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 M-BM-5g cRNA hybridized in duplicates for each of the three groups to the GeneChip arrays. Group1: naM-CM-/ve, Group2: liver-activated Group3: gut-activated. Lists of differentially regulated genes were created using High Performance Chip Data Analysis (HPCDA) with Bioretis database (http://www.bioretis-analysis.de). Worldwide data sharing is possible via Bioretis, please ask the authors.
Project description:In this experiment, the gene expression of CD8+ T cells primed in the presence or absence of Tregs on a single cell level was analyzed. For this purpose, Ly5.1 OT-I T cells were adoptively transferred into Treg deficient (DEREG+) or Treg replete (DEREG-) mice, activated with DC-OVA, isolated after 3 days, and analyzed by scRNAseq. Invididual samples were indexed with oligo-tagged TotalSeq-C antibodies and pooled prior to the analysis. We observed that Tregs reduced the expression of IL-2 responsive genes, including key cytotoxic molecule granzyme B. Unsupervised clustering revealed 5 clusters including a cluster of cells which did not match any usual CD8+ subsets and, due to unusual co-expression of effector molecules such as GZMK and KLRK1, were dubbed super-effector cells.
Project description:This study reports the improvement in function and antitumor efficacy of CXCR4 overexpression in therapeutic CD8+ T cells. Polyclonal CD8+ T cells from OT-I C57BL/6 (B6) mice were transduced with a modified pMP71 retroviral vector containing murine Cxcr4 and Gfp reporter sequences (T_CXCR4) or with a control vector containing Gfp alone (T_Control), and co-transferred into to B6 mice that were then vaccinated with peptide-pulsed DC. 90 days following DC vaccination, lymphocytes were isolated from the spleen and resting memory OT-I T_CXCR4 and OT-I T_Control cells were FACS sorted to perform gene expression profiling.
Project description:We analysed the chromatin opening and accessibillity of transcription factor binding motifs of in vitro activated OT-I CD8+ T cells in the absence of NR4A3 to determine the early impact of NR4A3 in CD8 T cell differentiation
Project description:Interleukin 6 (IL-6) is a pleiotropic cytokine with diverse roles in homeostasis, inflammation, and cancer. Here we performed transcriptional profiling of OT-I CD8+ T cells activated in the presence or absence of IL-6. In this study, splenocytes from wild type C57BL/6J mice were incubated with SIINFEKL peptide for two days to activate OT-I CD8+ T cells. SIINFEKL was then withdrawn, T cells were rested in medium with IL-2 for three days, and CD8+ T cells were restimulated with anti-CD3 + anti-CD28 antibodies and purified by FACS for RNA sequencing analysis on day 7. Throughout this culture period, some cells were treated with anti-IL6R antibody (clone MR16-1, 5 ug/ml), isotype control antibody (5 ug/ml), recombinant IL-6 (10 ng/ml), or recombinant hyper-IL-6 (IL-6 linked to IL6R; 20 ng/ml). Experimental conditions were run in triplicate, for a total of 15 samples. Conclusion: Neutralization of endogenous IL-6 signaling (with anti-IL6R antibody) enhances effector differentiation of CD8+ T cells, while addition of exogenous IL-6 or hyper-IL-6 suppresses effector differentiation.