title: Assessment of caspase mediated degradation of linker for activation of T cells (LAT) at a single cell level.
abstract: 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(+)CD8(low) thymocytes i.e. precursors of thymus derived CD4(+) regulatory T cells, in contrast to Foxp3(-)CD4(+)CD8(low) 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.
authors.affiliation: Laboratory of Tumor Immunology, Department of Tumor Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Mikołaj
authors.initials: M
authors.lastname: Kłossowicz
authors.forename: Bożena
authors.initials: B
authors.lastname: Scirka
authors.forename: Joanna
authors.initials: J
authors.lastname: Suchanek
authors.forename: Karolina
authors.initials: K
authors.lastname: Marek-Bukowiec
authors.forename: Pawel
authors.initials: P
authors.lastname: Kisielow
authors.forename: Enrique
authors.initials: E
authors.lastname: Aguado
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Miazek
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D020169:Caspases / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000939:Epitopes; C491734:Lat protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; D020169:Caspases