Analysis by flow cytometry of tyrosine-phosphorylated proteins in activated T-cell subsets on whole blood samples.

Tyrosine phosphorylation of cellular proteins is a critical early event in the T-cell activation process induced by the antigenic peptide or monoclonal antibodies specific for the CD3 T-cell receptor (TCR) complex. Phosphoproteins are currently detected by Western blotting experiments or, recently, by labelling intracellular proteins with an antiphosphotyrosine monoclonal antibody and flow cytometric analysis (Farahi Far et al.: Cytometry 15:327-334, 1994; Vuillier et al.: J Immunol Methods 185:43-56, 1995). Here, we describe improvements of these latter methods in order to study selectively the CD3-TCR signaling pathway of patients with immunodeficiency diseases or lymphopenia. This new technique quantifies tyrosine-phosphorylated proteins in in vitro-activated T-cell subsets directly on whole blood samples. The stimulation of the CD3-TCR complex induces a specific and significant increase in the phosphotyrosine fluorescence intensity in both CD4 and CD8 subpopulations. The simplicity and the good reproducibility of this method make it particularly convenient for laboratory routine evaluation, and the use of very small volumes of blood is well adapted to the study of immunodepressed patients. Moreover, this technique allows the detection of early molecular defects of the CD3-TCR signal-transduction pathway.