Assessment of caspase mediated degradation of linker for activation of T cells (LAT) at a single cell level

Caspase/Granzyme B mediated protein degradation is involved in elimination of activated T cell receptor (TCR) signaling molecules during processes of thymocyte selection and maintenance of peripheral homeostasis of T cells. Key components of TCR signaling cassette including LAT undergo biological inactivation in response to pro-apoptotic or anergy inducing environmental stimuli. Although available Western immunoblotting-based techniques are appropriate for detection of protein degradation in bulk populations of target cells, quantitative assessment of this process at a single cell level requires a different approach. Here we report on a novel, flow cytometry-based method for assessment of LAT integrity. This method exploits a loss of an anti-LAT antibody epitope recognition following proteolytic degradation of C-terminal domain of the LAT. We show that the LAT degradation precedes phosphatidylserine translocation to the outer leaflet of the plasma membrane and thus may constitute an early marker of T cell apoptosis. When used in conjunction with multi-parameter flow cytometry, our method revealed that FoxP3+CD4+CD8low thymocytes i.e. precursors of thymus derived CD4+ regulatory T cells, in contrast to Foxp3− CD4+CD8low thymocytes are resistant to LAT degradation in response to CD3ε crosslinking. This finding can be used as an additional marker for T regulatory cell lineage.