Core Fucosylation Of T Cell Receptor Is Crucial For Thymocytes Development At Double-positive(DP) Stage

Background:In thymus,T cell progenitors proceed through a series of cell proliferation and differentiation stages that are tightly controlled at multiple checkpoints associated with differential gene expression and regulation.Thymocytes undergo a complex pattern of differentiation characterized by sequential changes in expression of the cell surface markers CD4 and CD8 through the stages DN?CD4^-CD8^-Double-negative?,DP?CD4⁺CD8⁺Double-positive?,CD4 SP?CD4⁺CD8^-Single-positive?,and CD8 SP?CD4^-CD8⁺single-positive?.Early DN thymocytes undergo a complex pattern of differentiation characterized by sequential changes in expression of the cell surface markers CD25 and CD44 through the stages DN1?CD25^-CD44⁺?,DN2?CD25⁺CD44⁺?,DN3?CD25⁺CD44^-?,and DN4?CD25^-CD44^-?.TCR?T cell receptor?induces ligand independent activation signals for thymocyte survival,proliferation and differentiation.Thus TCR has a unique role in thymocyte development.TCRs are heavily core fucosylated glycoprotein.Core fucosylation mediated by Fut8?core fucosyltransferase?is an important post-translational process,and it has been shown to be necessary for the function and stability of cell-surface glycoproteins.Objective:We use Fut8^+/+and Fut8^-/-mice DP thymocytes to examine the impact of core fucosylation on T cell development,and to find core fucosylation of TCR is essential for normal thymocyte development at DP stage.Methods:Sorting the subpopulations of DN,DP,CD4⁺SP and CD8⁺SP cells from the thymus of Fut8^+/+mice at 4 to 8 weeks old,and then the cells were stained with LCA.We examined the expression of core fucosylation on the cell-surface by FACS?Fluorescence Activated Cell Sorter?.We separated thymocytes from Fut8^+/+and Fut8^-/-mice at 4 to 8 weeks old.After sorting of the subpopulations of DN1,DN2,DN3,DN4,DP,CD4⁺SP and CD8⁺SP cells,we calculated the number of cells.Then we explored the effect of the ablation of core fucosylation on the number of thymocytes in different stages of mice.We detected the difference of the core fucosylation of TCR on the surface of Fut8^+/+and Fut8^-/-DP cells via IP?Immunoprecipitation?,Western Blot and FACS.And we examined the level of phosphorylation by Western Blot.To further explore the role of core fucosylation in TCR signal transduction,Fut8^+/+OT-II or OT-I mice DP cells were treated with fucosidase,which can cleave?1,6-linked fucose more efficiently than other fucose linkages,and then we analyze the TCR signal transduction.Moreover,we detected the proliferation of DP cells by MTT assay and colony assay.The last we measured the expression of multiple transcription factors associated with TCR signaling by Q-PCR?Real-time Quantitative PCR Detecting System?.Results:Core fucosylation levels show dynamic changes during T cell development.These findings implied that Fut8 plays a crucial role in the stage of DN cells properly differentiate to DP cells.Ablation of Fut8 leads to reduced T cell numbers in thymus.Compared with Fut8^+/+DP cell,Fut8^-/-DP cell phosphorylation level and proliferation showed a striking decrease.The levels of TCR-mediated signal transduction and proliferation were significantly decrease,which treatment with fucosidase.And we found the expression of transcription factors of TCR signaling transduction have an abnormal expression in Fut8^-/-DP cellConclusion:Our results demonstrate that the core fucosylation plays a crucial role during thymus development.Core fucosylation of TCR is essential for normal thymocyte development at DP stage.