After overnight

α-CD3 stimulation, both TSC1KO CD4+ and C

After overnight

α-CD3 stimulation, both TSC1KO CD4+ and CD8+ T cells upregulated CD25 and CD69 in a heterogeneous manner. A portion of TSC1KO T cells showed decreased CD25 and CD69 upregulation as compared with WT T cells (Fig. 2F), suggesting impaired early activation of T cells in the absence of TSC1. α-CD3 stimulation resulted in expansion of WT CD4+ T cells in vitro. Such expansion appeared blunted in the absence of TSC1 (Fig. 2G). However, TSC1KO CD4+ as well as CD8+ T cells underwent similar or even more divisions than WT T cells during the same time of α-CD3 stimulation (Fig. 2H). Although a decrease in CD4+ T-cell expansion was observed, elevated levels of IL-2 were detected in the supernatants of TSC1KO CD4+ T cells compared with that of WT CD4+ T cells after 48 or 72 h of stimulation with α-CD3 (Fig. 2I), suggesting increased IL-2 production by TSC1KO T cells on GSK1120212 cost JAK inhibitor a per cell basis. These results indicate that TSC1 deficiency results in constitutive activation of mTORC1 in thymocytes and peripheral T cells, and has complex effects on T-cell activation manifested by decreased early activation and blunted expansion, but increased

IL-2 production and slightly enhanced proliferation. The decreases in both CD4+ and CD8+ peripheral T-cell compartments in TSC1-deficient mice, and the blunted expansion concordant with normal or enhanced proliferation of TSC1KO T cells in vitro led us to hypothesize that TSC1 might control T-cell survival. Indeed, an increased proportion of freshly isolated TSC1KO CD4+ and CD8+ T cells stained positive for 7-AAD ex vivo (Fig. 3A). The increase in cell death of TSC1KO T cells was not associated with the upregulation of Fas or FasL (Fig. 3B). The vast majority of cell death within the T-cell subsets is confined to the CD44hiCD62Llow population in both WT and TSC1KO T cells, and the death occurring in this particular subset is noticeably pronounced NADPH-cytochrome-c2 reductase in TSC1KO T cells (Fig. 3C). The amount of cell death seen in TSC1KO T cells was intensified after α-CD3

stimulation (Fig. 3D). Collectively, these observations demonstrate that the absence of TSC1 in T cells renders them less fit for survival in the periphery, particularly during T-cell activating conditions. The mitochondrion plays a central role in apoptosis 22. In HSCs, TSC1-deficiency results in increased mitochondrial content and the production of harmful ROS 18. To our surprise, TSC1KO T cells exhibited decreased mitochondrial content compared with WT T cells based on MitoTracker Green staining (Fig. 4A). Also, the ratio of mitochondrial DNA to nuclear DNA using the 12S rRNA gene and 18S rRNA as mitochondrial and nuclear DNA markers, respectively, was significantly decreased in TSC1KO T cells (Fig. 4B).

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