Summary
The reason(s) why individual cytotoxic T lymphocytes (CTL) possess a fast‐acting, perforin/granzyme‐mediated, as well as a much slower, Fas ligand (FasL) ‐driven killing mechanism is not ...clear, nor is the basis for wide variations in killing activity exhibited by individual CTL, ranging from minutes to hours. We show that perforin expression among individual, conjugated CTL varies widely, which can account for the heterogeneity in killing speeds exhibited by individual CTL. Despite a 2‐hr lag in FasL‐based killing, CTL lytic action is enhanced when the two mechanisms operate in concert. This is explained by finding that the two pathways in fact are jump‐started simultaneously with the lag in FasL lytic action reflecting pre‐lytic caspase‐8 activation and BH3‐interacting domain (BID) cleavage. The complementary action of the two lytic pathways, co‐expressed at varying levels among individual CTL, facilitates the lytic action of late‐stage poor perforin‐expressing CTL, ensuring optimal cytocidal action throughout the CTL response.
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein ...binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
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•Meta-analysis of human, non-human primate, and mouse single-cell RNA-seq datasets for putative SARS-CoV-2 targets•Type II pneumocytes, nasal secretory cells, and absorptive enterocytes are ACE2+TMPRSS2+•Interferon and influenza increase ACE2 in human nasal epithelia and lung tissue•Mouse Ace2 is not upregulated by interferon, raising implications for disease modeling
Analysis of single-cell RNA-seq datasets from human, non-human primate, and mouse barrier tissues identifies putative cellular targets of SARS-CoV-2 on the basis of ACE2 and TMPRSS2 expression. ACE2 represents a previously unappreciated interferon-stimulated gene in human, but not mouse, epithelial tissues, identifying anti-viral induction of a host tissue-protective mechanism, but also a potential means for viral exploitation of the host response.
Advances in molecular cell biology, medical research, and drug development are driving a growing need for technologies that enable imaging the dynamics of molecular and physiological processes ...simultaneously in numerous non-adherent living cells. Here we describe a platform technology and software--the CKChip system--that enables continuous, fluorescence-based imaging of thousands of individual living cells, each held at a given position ("address") on the chip. The system allows for sequential monitoring, manipulation and kinetic analyses of the effects of drugs, biological response modifiers and gene expression in both adherent and non-adherent cells held on the chip. Here we present four specific applications that demonstrate the utility of the system including monitoring kinetics of reactive oxygen species generation, assessing the intracellular enzymatic activity, measuring calcium flux and the dynamics of target cell killing induced by conjugated cytotoxic T-lymphocytes. We found large variations among individual cells in the overall amplitude of their response to stimuli, as well as in kinetic parameters such as time of onset, initial rate and decay of the response, and frequency and amplitude of oscillations. These variations probably reflect the heterogeneity of even cloned cell populations that would have gone undetected in bulk cell measurements. We demonstrate the utility of the system in providing kinetic parameters of complex cellular processes such as Ca++ influx, transients and oscillations in numerous individual cells. The CKChip opens up new opportunities in cell-based research, in particular for acquiring fluorescence-based, kinetic data from multiple, individual non-adherent cells.
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