Project description:T cells from spleen and lymphnode were isolated with EasySep Mouse CD8+ T Cell Isolation kit. Isolated CD8+ T cells were stimulated and cultured with plate-bound anti-CD3 and soluble anti CD28 antiboies in Tc1 or Tc9 cell polarizing conditios. After 3-day of polarization, cell were transferred into a new well and culture for another 2-day. on day 5 after seeding, Tc1 and Tc9 cells were collected and sequenced. We used microarrays to detail the global programme of gene expression of Tc1 and Tc9 cells at day 5 polarization.
Project description:The goal of this project is to dissect the mechanism by which Tc1 and Tc2 effector subtypes effect cell physiology in vitro. I utilized cellular proteomics to determine Tc1 and Tc2 secretion patterns and identify inflammatory factors that regulate cell polarization and physiology in vitro.
Project description:we use RNA sequence to detect the global gene expression in tumor-infiltrating Tc1 and Tc9 cells after being adoptively transferred in vivo
Project description:We provide an original multi-stage approach identifying a gene signature to assess the fibroblast polarization. Prototypic polarizations (inflammatory/fibrotic) were induced by seeded mouse embryonic fibroblasts (MEFs) with TNFα or TGFß1, respectively. The transcriptomic and proteomic profiles were obtained by RNA microarray and LC/MS-MS. Gene Ontology and pathways analysis were performed among the differentially expressed genes (DEGs) and proteins (DEPs). Balb/c mice underwent daily intradermal injections of HOCl (or PBS) as an experimental murine model of inflammation-mediated fibrosis in a time-dependent manner. As results, 1,456 and 2,215 DEGs, and 289 and 233 DEPs were respectively found in MEFs in response to TNFα or TGFß1, respectively. Among the most significant pathways, we combined 26 representative genes to encompass the proinflammatory and profibrotic polarizations of fibroblasts. Based on principal component analysis, this signature deciphered baseline state, proinflammatory polarization, and profibrotic polarization as accurately as did RNA microarray and LC/MS-MS. Then, we assessed the gene signature on dermal fibroblasts isolated from the experimental murine model. We observed a proinflammatory polarization at day 7, and a mixture of a proinflammatory and profibrotic polarizations at day 42 in line with histological findings. Our approach provides a small-size and convenient gene signature to assess murine fibroblast polarization.
Project description:The triggers that drive interferon-g (IFNg)-producing CD8 T cell (Tc1 cell)-mediated autoimmune hepatitis (AIH) remain obscure. Here we show that lack of hematopoietic Tet methylcytosine dioxygenase 2 (Tet2), an epigenetic regulator associated with autoimmunity, results in the development of microbiota-dependent AIH-like pathology, accompanied by hepatic enrichment of aryl hydrocarbon receptor (AhR) ligand-producing pathobionts and rampant Tc1 cell immunity. We report that AIH-like disease development is dependent on both IFNg and AhR signaling, as blocking either reverts ongoing AIH-like pathology. Illustrating the critical role of AhR ligand-producing pathobionts in this condition, hepatic translocation of the AhR ligand indole-3-aldehyde (I3A)-releasing Lactobacillus reuteri is sufficient to trigger AIH-like pathology. Finally, we demonstrate that I3A is required for L. reuteri-induced Tc1 cell differentiation in vitro and AIH-like pathology in vivo, both of which are restrained by Tet2 within CD8 T cells. This AIH-disease model may contribute to the development of therapeutics to alleviate AIH.