ABSTRACT Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T-cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8 + T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8 + T cells on the synthesis versus uptake of various non-essential amino acids during T-cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T-cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase ( Asns ), whose expression sharply peaks for effector CD8 + T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro , by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8 + T-cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8 + T-cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8 + T-cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.
Background Plasminogen-deficient mice display impaired vascular wound healing and reduced arterial neointima formation after arterial injury, suggesting that inhibition of plasmin generation might reduce arterial neointima formation. Therefore, we studied the consequences of plasminogen activator inhibitor-1 (PAI-1) gene inactivation and adenoviral PAI-1 gene transfer on arterial neointima formation. Methods and Results Neointima formation was evaluated in PAI-1–deficient (PAI-1 −/− ) mice with perivascular electric or transluminal mechanical injury. PAI-1 deficiency improved vascular wound healing in both models: the cross-sectional neointimal area was 0.001±0.001 mm 2 in PAI-1 +/+ and 0.016±0.008 mm 2 in PAI-1 −/− mice within 1 week after electric injury ( P <.02) and 0.055±0.008 mm 2 in PAI-1 +/+ and 0.126±0.006 mm 2 in PAI-1 −/− mice within 3 weeks after mechanical injury ( P <.001). Proliferation of smooth muscle cells was not affected by PAI-1 deficiency. Topographic analysis of arterial wound healing after electric injury revealed that PAI-1 −/− smooth muscle cells, originating from the uninjured borders, more rapidly migrated into the necrotic center of the arterial wound than wild-type smooth muscle cells. On the basis of immunostaining, PAI-1 expression was markedly upregulated during vascular wound healing. There were no genotypic differences in reendothelialization of the vascular wound. When PAI-1 −/− mice were intravenously injected with replication- defective adenovirus expressing human PAI-1 (AdCMVPAI-1), plasma PAI-1 antigen levels increased in a dose-dependent fashion up to to 61±8 μg/mL with 2×10 9 plaque-forming units (pfu) virus. Luminal stenosis was 35±13% in control AdRR5-treated (2×10 9 pfu) and suppressed to 5±5% in AdCMVPAI-1–treated (6×10 8 pfu) PAI-1 −/− mice ( P <.002). Conclusions By affecting cellular migration, PAI-1 plays an inhibitory role in vascular wound healing and arterial neointima formation after injury, and adenoviral PAI-1 gene transfer reduces arterial neointima formation in mice.
Many studies suggest that the plasminogen activator (PA) system plays a role in the proteolytic degradation of the follicle wall at the time of ovulation. Consistently, the ovulation efficiency is reduced by 26% in mice where both physiological PA genes have been inactivated. To reveal the mechanism behind reduced ovulation efficiency in PA‐deficient mice and its effect on ovarian proteolysis, we have studied the regulation of plasmin activity in the ovaries of 25‐day‐old wild‐type mice and mice with deficient PA gene function during gonadotropin‐induced ovulation. In wild‐type mice the plasmin activity was low in ovarian extracts from mice treated with pregnant mare's serum gonadotropin. However, this activity was increased between 2–8 h after an ovulatory dose of human choriogonadotropins. In mice lacking either tissue‐type PA (tPA) or PA inhibitor type 1 (PAI‐1) the plasmin activity levels prior to ovulation were similar to wild‐type mice, while extracts prepared from urokinase‐type PA (uPA) deficient mice had 10% or less of the plasmin activity. This indicates that most of the plasmin activity in the mouse ovary is generated by uPA. In addition, as the ovulation efficiency is impaired in tPA/uPA‐deficient mice but appears normal in uPA‐deficient mice, our data indicates that the amount of plasmin generated by PAs prior to ovulation in wild‐type mice greatly exceeds the amount required for efficient ovulation.
