Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation

Using human CD4(+) T-cell clones and peptide-pulsed antigen-presenting cell s (APC) we measured, at the single cell level, different steps in the T-cel l activation cascade. Simultaneous analysis of T-cell antigen receptor (TCR ) down-regulation and interferon-gamma (IFN-gamma) production shows that bo th the level of TCR occupancy and the amount of IFN-gamma produced by singl e T cells increase in an antigen dose-dependent fashion. Conversely, commit ment of T cells to IFN-gamma production does not occur as soon as a defined number of TCR have been engaged, but requires the same duration of sustain ed signalling at low as well as at high antigen concentrations. Measurement of phosphotyrosine levels by flow cytometry reveals that, upon conjugation with APC, individual T cells undergo an antigen dose-dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupan cy. In antigen-stimulated T cells the increased phosphotyrosine staining is localized in the area of contact with APC, as shown by confocal microscopy . PTK activation is sustained for at least 2 hr after conjugation, and is r equired to maintain a sustained increase in intracellular Ca2+ concentratio n. Our results show, for the first time, a direct correlation between the l evel of TCR occupancy and the activation of PTK in individual T cells and o ffer an explanation for how the number of triggered TCR can be 'counted' an d integrated in a corresponding biological response.