| Research Abstract |
This project aimed to analyze the molecular mechanism of antigen peptide/MHC recognition on antigen-presenting cells and activation by T cells at single molecule level. We introduced three improvements in the system for this purpose ; first, we utilized the planar membrane system containing GPI-anchored MHC and ICAM-1 molecule instead of antigen-presenting cells. Second, we used normal T cells from TCR-transgenic mice into which fluorescence-labeled signaling molecules were introduced by retrovirus. Third, a sensitive microscopy system, TIRF (total internal reflection fluorescence) was used to detect the movement at single molecule level. We found that TCR microclusters were generated upon antigen stimulation and induced activation signals. From the analysis at single molecule level, we found that a single microcluster contains roughly 50-300 TCR molecules accumulated at the early activation, and they moved towards the center of Immunological synapse. At later time point after c-SMAC w
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as completed, microclusters were newly generated at the periphery and functioned as the site for generating sustained activation signals. We analyzed a series of signaling molecules accumulated within microclusters, and found that Lck, LAT, SLP-76, PLCγ, Vav, PI3K contributed to generate microclusters at early time upon antigen stimulation. On the other hand, molecules involved in the activation in Ras and NF-κB including MEK, Erk, Ras, Carma-1, Bcl-10 are not accumulated in microclusters, and are not involved for direct activation. Furthermore, from the analysis of co-stimulatory molecules critical for T cell activation, we showed that the major costimulation receptor, CD28 is also accumulated into TCR microclusters and may function to recruit PKCθ in the presence of the ligand CD80. It has been known for a long time that PKCθ is a representative molecule accumulated into c-SMAC, but there is no mechanism of PKCθ recruitment. We suggested for the first time that it is regulated by co-stimulation. Less
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