FLOW CYTOMETRIC ANALYSIS OF PROTEIN-TYROSINE PHOSPHORYLATION IN PERIPHERAL T-CELL SUBSETS - APPLICATION TO HEALTHY AND HIV-SEROPOSITIVE SUBJECTS

This paper describes an improved method for detecting tyrosine phospho rylation levels in T cell subsets by flow cytometry early after CD3 cr osslinking stimulation. It consists in introducing gentle paraformalde hyde fixation between CD3 crosslinking in cold conditions and the shif t to 37 degrees C, which activates downstream signalling machinery. We used the combined properties of monoclonal antibodies for stimulating cells and for detecting surface markers to analyze protein-tyrosine p hosphorylation levels in T cell subsets following stimulations which m imic physiological activation. Overall data obtained in healthy subjec ts, using two-or three-color immunofluorescence, indicated that: (1) m ost CD3 positive cells phosphorylate tyrosine substrates following CD3 crosslinking stimulation and (2) helper cells phosphorylate tyrosine to a slightly better extent than cytotoxic cells after CD3 crosslinkin g. Nevertheless, the two subsets follow similar kinetic patterns and t end to retain a homogeneous profile. Processing of samples from HIV-se ropositive patients showed heterogeneous phosphorylation levels in bot h subsets, when compared to normal donors. This assay should, in the f uture, lead to easy and rapid exploration of the signal transduction p athway in different subsets of T cells under normal and pathological c onditions.