The rapid and efficient phagocytic clearance of apoptotic cells by efferocytosis is a critical process for maintaining tissue homeostasis and immune function. T cells undergo apoptosis during thymic education and in the periphery, and require rapid clearance. CD43 is a bulky and heavily-glycosylated transmembrane protein highly expressed on the T cell surface. The function of CD43 in immune responses is mostly attributed to its anti-adhesive properties that negatively regulate cell-cell interactions. However, how CD43 might be modulated during cell apoptosis and whether such potential modulation might influence efferocytosis are not known. In this study, I demonstrate that CD43 is promptly and selectively eliminated from the human T cell surface during apoptosis, and the removal is mediated by the ADAM10 sheddase, whose activation is associated with surface-exposed phosphatidylserine that flips from the inner to outer leaflet of the plasma membrane. I established a novel efferocytosis assay and used this to show that CD43 removal increases the uptake of apoptotic T cells in an ADAM10-regulated manner. These findings present a novel regulatory pathway for CD43 expression and reveal CD43 as a ‘don’t eat me’ barrier for efferocytosis with implications for developing therapeutic strategies against pathological conditions that are associated with efferocytosis.

Zika virus (ZIKV) is a pathogen that displays neurotropism for the fetal brain particularly during early trimesters. To date, research showing metabolic dysregulation in brain cells infected with ZIKV suggested this to be a potential cause of ZIKV-fetal microcephaly. Herein, we used the high-content power of Imagestream to investigate metabolic dysregulations during ZIKV infection. To do this, we analysed several features of cellular metabolism such as size and compactness of mitochondria and neutral lipids in infected and neighbouring non-infected neuronal progenitors. We showed that ZIKV reduces mitochondrial size and number in a bystander fashion with a direct correlation between infection stage and neuronal maturation. To corroborate these findings, we then tested metabolic modulating drugs that exert differential impact on mitochondrial biogenesis and fusion-fission processes. Dimethyl fumarate (DMF) and dichloroacetate (DCA) dampened ZIKV titers in our models of early mitochondrial dysregulation whilst in our models of late mitochondrial dysregulation, only DCA dampened ZIKV titers. These results potentially suggest specific mitochondrial changes in neuronal cells over different trimesters and thus are of consideration for the development of metabolic therapies against ZIKV. Furthermore, we showed that high-content imaging-based results obtained using Imagestream reliably identify mitochondrial dynamics with implications in the development of therapeutics in metabolic disorders.

What are the driving and limiting factors in mammalian cell growth, how is gene expression homeostasis preserved during the cell cycle and how do these relate to gene expression noise? To answer these questions, we use multiparametric imaging flow cytometry (Imagestream) to acquire a high resolution snapshot of the cell cycle, by sampling from an asynchronously growing cell population. Using computational trajectory reconstruction, we combine multiple fluorescent markers of the cell cycle into a continuous pseudo-time estimation. We use the Imagestream’s single cell brightfield images to obtain precise cell-size measurements, which in conjunction with the pseudo-time estimation reveal changes in growth rate throughout the cell cycle. Using metabolic labelling, we measure changes in transcription and translation rate and investigate how these correlate with changes in the cell growth rate. Finally, we use single-molecule Fluorescent In Situ Hybridisation (smFISH) to measure gene expression, and show that the ability of cells to maintain a constant concentration of mRNA transcripts varies drastically during the cell cycle.

The ImageStream® is a multispectral CCD-based imaging flow cytometer that helps overcome the obstacles of traditional flow cytometry by combining the statistical power of flow cytometry with the imaging content of microscopy in one system.
With the ImageStream®, we are now able to characterize all cell types including small particles, such as viruses and including EVs such as exosomes (ranging from 30 nm to 160 nm in diameter), microvesicles, and apoptotic bodies at the single EV level.
Moreover, due to its low shear forces, imaging capabilities, and combination with state-of-the-art AI for data analysis, the ImageStream® can be used to study rare cell-cell interactions and for the detection of CTCs. It prevents the misclassification of EpCAM+ CD45+ events as leukocytes and the undercounting of CTC doublets as single cells.
The Imagestream data is publishable and should be THE METHOD you should think of first for trouble-shooting difficult traditional flow data, and when trying to quantify images-based data in a way that takes the bias and subjectivity out of your data – See behind the dots and visually confirm your science.