Knockout CD45 in LatY136F mice affact T cell activation
Background: The adaptor protein Linker for activation of T cell (LAT) is a key signaling hub used by the T cell antigen receptor. Mice with a mutation at the LAT-PLC?1 binding site (Y136) have a defect in thymocyte development due to dampened TCR signaling. CD4 + T cells that do reach the periphery are hyper-activated and skewed to Th2. Over time, these mice develop an autoimmune-like syndrome, characterize by overproduction of Th2 cytokines, T cell infiltration into various organs, and B cell activation, isotype switching, and autoantibody production. In this study, we established LatY136F and   LatY136F-CD45 KO  mice and using them to investigate to role of CD45 in  TCR signalling via flow cytometry and scRNA sequencing. Conclusion: Knockout CD45 in LatY136F mice affact T cell activation Overall design: For scRNA-seq analysis of T cells, 8-9 weeks old LatY136FCD45-KO and LatY136F mice were sacrificed. Single cell suspension from spleens were stained with antibody CD4-PE-cy7 and CD3-FITC . After washing with 1 mL FACS buffer two times, cells were resuspended with Sytox blue and CD3+CD5+ cells were sorted and subjected to the BD Rhapsody Express system. Then cDNA and sample tag libraries were built with BD Rhapsody whole transcriptome amplification (WTA) reagent kit (BD Bioscience, Cat: 633733, 633773, 633801, 664887) following manufacturer's instructions, and sequenced on an illumine Novaseq 600 sequencer. Pair-end Fastq files of sample tag and WTA data were processed via BD RhapsodyTM analysis pipline v2.0, and the resultant dataset was mainly analysis using SeGeqTM software, which contain Lex-BDSMK and Seurat v4.04 plugin components.