T-cell entosis in the liver: catching escaping t-cells and The expression of e3 ubiquitin ligases in t-cells

[摘要] Entosis describes a form of emperipolesis, where one cell invades a host cell. Emperipolesis can result in death, division or release of the internalised cell. Preliminary data demonstrates that entosis and release of CD4+T-cells occurs within primary hepatocytes and hepatoma cell lines.This study focuses on defining the environment and stimuli necessary to induce T-cell release following entosis. A 7-Day release assay was optimised for measuring CD4+T-cell release following T-cell-hepatoma co-culture and entosis. Flow cytometry was then used to quantify the release of T-cells from HepG2-CD81 and Huh-7 cell lines in response to various stimuli, doses and incubation times. Results indicated that the majority of stimuli and dose responses were not responsible for inducing a higher or lower %T-cell release. Co-cultures also demonstrated a significantly higher %T-cell release from HepG2-CD81s at 2 hour compared to 24 hour incubations in treated and untreated co-cultures. Following optimisation of this release assay, the study concludes that further work is required to investigate the conditions which induce entosis and release of T-cells from hepatoma cell lines. Understanding the purpose and cause of T-cell entosis and release in the liver may lead to its therapeutic manipulation to prevent liver inflammation and diseases such as hepatitis. E3 ubiquitin ligases have been identified as key regulators of the immune system. They function in T-cell tolerance by targeting molecules for degradation, thereby affecting T-cell signalling and activation. By understanding the role of E3 ubiquitin ligases in different T-cell subsets, we may be able to manipulate them therapeutically to stop or promote T-cell activation, thereby preventing development of autoimmune diseases and cancer respectively. This study induces anergy in human CD4+T-cells through plate-bound anti-CD3 antibody stimulation. Anergy in anti-CD3 stimulated cells was determined by quantifying the number of proliferating T-cells by flow cytometry following a secondary anti-CD3.28 stimulus. Quantitative RT-PCR was also used to establish differences in E3 ubiquitin ligase expression between T-cell subsets. Results showed some induction of anergy in anti-CD3 stimulated T-cells. When comparing E3 ubiquitin ligase expression between different T-cell subsets, many of the genes were not detectable. This may be due to large amounts of cell death during assays resulting in low cDNA recovery and insufficient material to detect gene expression. This study concludes that further work is required to confirm the anergic state of the T-cells. Furthermore, in order to accurately compare E3 ligase expression between T-cell subsets, RT-PCR needs to be repeated using more material.

[发布机构] University:University of Birmingham;Department:School of Immunity and Infection

[关键词] R Medicine;RK Dentistry