T cell receptor signal transduction involved in T cell anergy

1)免疫受体酪氨酸基激活基序（Immunoreceptor tyrosine-based activation motif， ITAM）由两个YxxL片段组成，在T细胞中传递导致IL-2基因激活的信号。我们研究了这两个YxxL片段在itam介导的信号转导中的功能差异。即使存在CD28共刺激，n端YxxL突变体也不能诱导ZAP70磷酸化、细胞内Ca2^+浓度升高（[Ca2^+]i）或细胞外信号调节激酶（ERK）激活，而c端YxxL片段亮氨酸残基突变体保留了诱导这些事件的能力，尽管这种突变消除了诱导IL-2基因激活的能力。与ERK活化形成鲜明对比的是，当与CD28共刺激时，所有突变体都观察到c-Jun nh2末端激酶（INK）活化。c端YxxL段亮氨酸残基突变体在NF-AT顺式元件的转录激活上存在缺陷，通过钙离子载体的加入使其恢复到野生型水平，表明[Ca2+]i升高的强度和/或持续时间决定了该突变体T细胞激活的阈值。我们的数据共同表明，ERK， JNK和钙动员的激活途径通过itam的YxxL片段进行差异调节。2)用特定对的抗cd2抗体刺激可以诱导T细胞活化和增殖。由于已经提出CD2参与T细胞能量，我们研究了ZAP70在CD2介导的信号转导中的作用。我们利用来自CDS缺陷患者的ZAP-70缺陷T细胞研究了ZAP-70在CD2信号传导中的意义，并表明ZAP-70是CD2刺激下T细胞增殖和细胞因子产生所必需的。生化分析显示，CD2刺激诱导ZAP-70缺陷T细胞中有丝裂原活化蛋白激酶（MAPK）超家族的激活，表明gdi触发的人T细胞中存在一个独立于ZAP-70的MAPK超家族激活途径。相比之下，细胞内Ca2+动员和活化T细胞核因子（NFAT）在CD2触发时的活化在这些T细胞中不存在。此外，我们发现药理学Ca2^+升高联合CD2刺激恢复了NFAT激活和随后的ZAP-70缺陷T细胞的细胞因子产生。这些结果表明，在CD2信号传导中，ZAP-70在Ca2^+动员和NFAT激活中起重要作用。少

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