The role of the adapter protein TSAd in T cell activation
Introduction: T cells are a part of the adaptive immune system, which is designed to attack pathogens in the body. T cells recognize antigen presented by HLA molecules on the surface of antigen presenting cells (APC) through their T cell receptor. Conjugation formation between T cells and APC turns on a system of intracellular events that eventually activates T cells. These intracellular events can lead to the activation of the transcription factor NF-κB. Upon activation, NF-κB translocates to the nucleus where it induces transcription of target genes. Adapter proteins are crucial in regulating intracellular signaling by promoting protein-protein interactions during signal transduction cascades but lack catalytic activity. T cell-specific adapter protein, TSAd, is expressed in activated T cells. TSAd interacts with the tyrosine kinases Lck and Itk and modulates their activity. These kinases are critical for the activation of T cells, however, the consequences of TSAd´s modulation of their activities are poorly understood. The aims of this study are: 1. Establish method for NF-κB translocation in activated T cells. 2. Establish and test conjugation of APC and transfected T cells, with or without TSAd. 3. Assess translocation of NF-κB in T cells forming conjugates with APC. Materials and methods: Experiments were done using Jurkat TAg cells (T cell line) and Raji cells (B cell line). Transfection with electroporation was used to introduce different cDNA construct into the Jurkat cells. Conjugation of Jurkat cells and Raji cells was performed using SEE super antigen to induce conjugate formation and to activate the Jurkat cells. All analyses were based on data from flow cytometry and image based cytometry, as well as Western blotting. Results: By stimulating Jurkat TAg cells using PMA, NF-κB translocation was evident after one hour of stimulation. Conjugation of APC and transfected T cells was performed using superantigen stimulation. Using time titration, relative NF-κB translocation in T cells peaked after one hour of superantigen stimulation. Jurkat T cells in conjugates, expressing TSAd wt, showed an increase in relative NF-κB translocation with time, although higher percentage of T cells, expressing the TSAd mutant protein, showed translocation. Discussion: Using TSAd transfected cells and a novel image based flow cytometry technique, we found that TSAd did influence relative NF-κB translocation upon APC presentation in T cell blasts but this needs to be further examined to be able to clearly define the role of TSAd in the activation of NF-κB.
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