T cell receptor signal transduction involved in T cell anergy

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1) Immunoreceptor tyrosine-based activation motif (ITAM), consisting of two YxxL segments, transmits signals leading to IL-2 gene activation in T cells. We investigated the functional difference between these two YxxL segments in the ITAM-mediated signal transduction. N-terminal YxxL mutants failed to induce ZAP70 phosphorylation, elevation of intracellular Ca2^+ concentration ([Ca2^+]i) or extracellular signal-regulated kinase (ERK) activation even in the presence of CD28 co-stimulation, whereas a mutant of the leucine residue at the C-terminal YxxL segment retainedlhe ability to induce these events although this mutation abrogates the ability to induce IL-2 gene activation. In marked contrast to ERK activation, c-Jun NH2-terminal kinase (INK) activation was observed in all mutants when co-stimulated with CD28. The mutant of the leucine residue at the C-terminal YxxL segment had defect in the transcriptional activation at the NF-AT cis-element, which was restored to the wild type leve … More l by addition of calcium ionophore, suggesting that the intensity and/or duration of [Ca2+]i elevation defines the threshold of T cell activation in this mutant. Our data collectively indicate that the activation pathways of ERK, JNK and calcium mobilization are differentially regulated through YxxL segments of an ITAM.2) Stimulation with specific pairs of anti-CD2 antibodies can induce T cell activation and proliferation. Since involvement of CD2 in T cell anergy has been proposed, we examined the role of ZAP70 in CD2-mediated signal transduction. We investigated the significance of ZAP-70 in CD2 signaling using ZAP-70 deficient T cells derived from a CDS deficient patient and showed that ZAP-70 is necessary for cellular proliferation and cytokine production in T cells stimulated via CD2. Biochemical analyses showed that CD2 stimulation induced activation of mitogen-activated protein kinase (MAPK) super family in ZAP-70 deficient T cells, indicating that a ZAP-70 independent pathway(s) exists for MAPK super family activation in GDI-triggered human T cells. In contrast, intracellular Ca2+ mobilization and activation of nuclear factor of activated T cells (NFAT) upon CD2 triggering were absent in these T cells. Furthermore, we found that pharmacological Ca2^+ elevation combined with CD2 stimulation restored NFAT activation and subsequent cytokine production in ZAP-70 deficient T cells. These results indicate that in CD2 signaling, ZAP-70 plays an essential role in Ca2^+ mobilization and NFAT activation. Less