CD3ε of T cell antigen receptor complex (TCR/CD3) plays an important role in the resembling of the complex and activation signaling through its conservative immunoreceptor tyrosine-based activation motif (ITAM) in th...CD3ε of T cell antigen receptor complex (TCR/CD3) plays an important role in the resembling of the complex and activation signaling through its conservative immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail. Previous study showed that a chimera molecule, consisting of the extracellular-transmembrane domain of human CD8α fused to the cytoplasmic domain of CD3ε, induced apoptosis of T lymphocytes, indicating that apoptotic signals were transduced through the CD3ε- ITAM. To elineate involvement of the two tyrosines in apoptotic signaling pathway, cDNAs with mutations at Y170F, Y181F and Y170F/Y181F in CD8-ε-ITAM were made by point mutation and PCR, and then cloned into pcDNA3 eukaryotic expression vectors. Stable expression cell lines were established after transfection of the expression vectors into CD8+- Jurkat T lymphocytes. Stimulation of these cell lines with anti-CD8 monoclonal antibody showed that only the cells with expression of wild type chimera CD8-ε died by apoptosis, but not those cells with expressions of mutated CD8-ε chimera, indicating that the two tyrosines in CD3ε-ITAM were required for the apoptotic signal transduction in T lymphocytes.展开更多
文摘CD3ε of T cell antigen receptor complex (TCR/CD3) plays an important role in the resembling of the complex and activation signaling through its conservative immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail. Previous study showed that a chimera molecule, consisting of the extracellular-transmembrane domain of human CD8α fused to the cytoplasmic domain of CD3ε, induced apoptosis of T lymphocytes, indicating that apoptotic signals were transduced through the CD3ε- ITAM. To elineate involvement of the two tyrosines in apoptotic signaling pathway, cDNAs with mutations at Y170F, Y181F and Y170F/Y181F in CD8-ε-ITAM were made by point mutation and PCR, and then cloned into pcDNA3 eukaryotic expression vectors. Stable expression cell lines were established after transfection of the expression vectors into CD8+- Jurkat T lymphocytes. Stimulation of these cell lines with anti-CD8 monoclonal antibody showed that only the cells with expression of wild type chimera CD8-ε died by apoptosis, but not those cells with expressions of mutated CD8-ε chimera, indicating that the two tyrosines in CD3ε-ITAM were required for the apoptotic signal transduction in T lymphocytes.
