Triggering of the T cell receptor (TCR) leads to the production of intracellular intermediates with half-life of a few minutes. Signaling kinetics of events originating from serial TCR triggering and its relation to antigen dose was investigated. In this study we documented incremental accumulation of short-lived intermediates of the extracellular signal-regulated kinase (ERK) family, produced during successive TCR triggering. The rate and extent of the intermediate accumulation are essentially determined by the level of TCR engagement and are augmented by costimulation. ERK-1 and ERK-2 exhibit different rates of accumulation following serial receptor triggering. The data indicate that the quantitative kinetic differences in downstream signaling pathways induce qualitatively distinct biological outcomes. Although CD69, interleukin-2, and interferon-␥ (IFN-␥) were primarily produced by high antigen doses that supported high MAPK phosphorylation, maximal interleukin-5 expression is induced by low and intermediate stimulus doses that do not support significant accumulation of activated ERK. We further demonstrated that the rate of phosphorylated ERK accumulation correlates with the duration of delay between T cell stimulation and the onset of IFN-␥ response, with stronger stimuli giving a more rapid IFN-␥ response. This delay might reflect the time required for the accumulation of signaling intermediates up to a threshold level that is necessary for activation. Thus, the data suggest that signaling events originating from serially triggered TCR are not simply sustained but are gradually accumulated and are integrated in a corresponding response.A single peptide antigen-major histocompatibility complex (MHC) 1 complex on the surface of an antigen-presenting cell (APC) can serially engage and trigger up to ϳ200 T cell receptors (TCRs) on a responding T cell (1). As a consequence of this "antigenic efficiency," relatively small numbers of peptide-MHC complexes on APC surfaces can trigger the threshold numbers of TCRs that are required for T cell activation (2).This intriguing feature of the T cell activation process makes physiological sense, in face of the antigenic complexity, as well as the consequent low frequency of any particular antigen, on the surface of any given APC in vivo.Any explanation of how serially receptor triggering leads to T cell activation must reconcile two opposing kinetic features. On the one hand, the signal emanating from each triggered TCR is short-lived and, in the absence of a continuous TCR triggering, the signal will stop. This point has been established by the demonstration that interference with continuous TCR engagement by peptide-MHC complexes can terminate signaling within minutes (3, 4). Furthermore, productive TCR engagement results in rapid TCR internalization and degradation (reviewed in Ref. 5). However, on the other hand, prolonged intracellular signaling over several hours is required for T cell activation (6).The intracellular signaling events underlying the serial trigge...

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TCR signal transduction is amplified by the dynamic accumulation of accessory molecules at APC-T cell contact sites, along with the simultaneous exclusion from these sites of negative regulators, such as certain tyrosine phosphatases and large glycosylated proteins. However, given the general nature of the cytoskeleton-driven clustering mechanism underlying molecular segregation events at the APC-T cell interaction site, the possibility exists that negative regulators might similarly be segregated at these sites. Using fluorescence microscopy, we have demonstrated that placental protein 14 (PP14), a direct T cell inhibitor, focuses toward APC-T cell contact sites in conjunction with conjugate formation. We have further established that the function of PP14 is dependent upon its localization to the sites of TCR triggering, where it negatively regulates T cell activation. Thus, PP14 provides an example of a soluble negative T cell regulator whose inhibitory activity is linked to modulation of the APC-T cell contact site, thereby hindering early events triggered by the TCR.