RNA-seq analysis of WT, CD5 KO, CD6 KO, CD5/CD6 double KO (I) and LAT KO naïve CD4+ T cells (II)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE148721 | GEO | 2020/09/29
REPOSITORIES: GEO
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Project description:RNA-seq analysis of WT, CD5 KO, CD6 KO, CD5/CD6 double KO (I) and LAT KO naïve CD4+ T cells (II)
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Project description:Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are upregulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We find that CD5 alters the basal activity of the NF-kB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NFκB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NFκB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This post-translationally stabilized cellular NFkB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide induced TCR signals lead to the upregulation of CD5, which subsequently maintains a proportional reserve of NFκB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.
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Project description:We report the differential gene expression between anti-CD3/CD28 stimulated WT and LRCH1-deficient CD8+ T cells. Improving cytotoxicity, proliferation and infiltration ability of CD8+ T cells is critical in T cell immunotherapies against tumors. This study has identified LRCH1 as a negative regulator of LAT-mediated TCR signal transduction, as LRCH1 inhibits LAT signalosome formation and facilitates the endocytosis of LAT on the plasma membrane. LRCH1-deficient CD8+ T cells are more proliferative and effective at pathogen control and tumor elimination. Importantly, CRISPR/Cas9-mediated knockout of LRCH1 in human T cells also increases IFN-γ production, cell proliferation and migration ability in vitro. These data suggest LRCH1 as a potential target to improve CD8+ T cell responses against tumor and pathogens.
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Project description:CD5 is characterized as an inhibitory co-receptor with important regulatory role during T cell development. To study the molecular mechanism by which CD5 operates, we used quantitative mass spectrometry to analyze the components of the CD5 signaling machinery in primary T cells. In a first set of experiments, CD5-containing complexes were immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice. Thymocytes were treated with pervanadate to induce widespread activation of protein tyrosine kinases. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls, to discriminate CD5-binding molecules from the background of contaminant proteins. Eight biological replicates were prepared for both conditions, and samples were analyzed in duplicate LC-MS runs. This first set of experiment is presented in TableS1 of the paper, and is associated to the MaxQuant result file “Interactome WT” in the present PX dataset. We analyzed the CD5 interactome at different time of stimulation with pervanadate (1min and 10min) using thymocytes from wild-type (WT) C57BL/6 mice. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls. Three biological replicates were prepared for both conditions (WT and KO) and each time point (1min and 10min). This set of experiments is presented in TableS2 of the paper and is associated to the MaxQuant result file “Interactome at 1min and 10min” in the present PX dataset. We checked that the activation of CD5 (phosphorylation sites) and the formation of the signaling complex was comparable in different stimulatory conditions (comparison of WT thymocytes stimulated with either pervanadate or anti-CD3+anti-CD4 antibodies). We also analyzed the binding of CD5 interactors in thymocytes from a c-Cbl-/- mouse model (comparison of WT and c-Cbl-/-thymocytes stimulated with pervanadate). These experiments and the associated experimental design of the comparisons are summarized in TableS3 of the paper, and are based on the MaxQuant result file “Comparison of different stimulatory conditions” in this dataset. To check the phosphorylation status of the CD5 tyrosine residues at different time of stimulation, we analyzed samples immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice, following stimulation with anti-CD3+anti-CD4 antibodies for 1min, 3min and 10min. This set of experiment is presented in TableS4 of the paper and is associated to the MaxQuant result file “CD5 pY kinetics” in the present PX dataset. To determine the role of Y429 in the context of CD5 signaling, we expressed a wild-type (CD5tgWt) or a mutated form of CD5, containing a tyrosine to phenylalanine substitution at position 429 (CD5tgY429F) in transgenic mice. We analyzed the CD5 interactome by comparing samples immunoprecipitated from thymocytes of these 2 mice models (following pervanadate stimulation, 6 and 5 biological replicates respectively) with samples prepared in the same way from Cd5−/− mice as controls (6 biological replicates). The results are shown in TableS5 of the paper, and correspond to the MaxQuant result file “interactome CD5tgWt_CD5tgY429F”.
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