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Recently, there has been an increasing emphasis on single cell profiling for high-throughput screening workflows in drug discovery and life sciences research. However, the biology underpinning these screens is often complex and is insufficiently addressed by singleplex assay screens. Traditional single cell screening technologies have created powerful sets of 'omic data that allow users to bioinformatically infer biological function, but have as of yet not empowered direct functional analysis at the level of each individual cell. Consequently, screening campaigns often require multiple secondary screens leading to laborious, time-consuming and expensive workflows in which attrition points may not be queried until late in the process. We describe a platform that harnesses droplet microfluidics and optical electrowetting-on-dielectric (oEWOD) to perform highly-controlled sequential and multiplexed single cell assays in massively parallelised workflows to enable complex cell profiling during screening. Soluble reagents or objects, such as cells or assay beads, are encapsulated into droplets of media in fluorous oil and are actively filtered based on size and optical features ensuring only desirable droplets ( e.g. single cell droplets) are retained for analysis, thereby overcoming the Poisson probability distribution. Droplets are stored in an array on a temperature-controlled chip and the history of individual droplets is logged from the point of filter until completion of the workflow. On chip, droplets are subject to an automated and flexible suite of operations including the merging of sample droplets and the fluorescent acquisition of assay readouts to enable complex sequential assay workflows. To demonstrate the broad utility of the platform, we present examples of single-cell functional workflows for various applications such as antibody discovery, infectious disease, and cell and gene therapy.

Cytotoxic T lymphocytes (CTLs) are key effector cells in the immune response against viruses and cancers, killing targets with high precision. Target cell recognition by CTL triggers rapid polarization of intracellular organelles toward the synapse formed with the target cell, delivering cytolytic granules to the immune synapse. Single amino acid changes within peptides binding MHC class I (pMHCs) are sufficient to modulate the degree of killing, but exactly how this impacts the choreography of centrosome polarization and granule delivery to the target cell remains poorly characterized. Here we use 4D imaging and find that the pathways orchestrating killing within CTL are conserved irrespective of the signal strength. However, the rate of initiation along these pathways varies with signal strength. We find that increased strength of signal leads to an increased proportion of CTLs with prolonged dwell times, initial Ca2+ fluxes, centrosome docking, and granule polarization. Hence, TCR signal strength modulates the rate but not organization of effector CTL responses., (© 2021 Frazer et al.)

Introduction: T cell and NK cell cytotoxicity can be mediated via the perforin/granzyme system and Fas Ligand (FasL, CD178). FasL is synthesized as a type II transmembrane protein that binds its cognate receptor Fas (CD95). Membrane-bound FasL is expressed on the plasma membrane of activated lymphocytes and is the main form of FasL with cytotoxic activity, but whether FasL is delivered to the immune synapse along with granzyme and perforin-containing granules is unclear., Methods: We stably expressed FasL-fluorescent fusion proteins into human NK cells and examined the localization of FasL relative to other intracellular markers by confocal and immunoelectron microscopy, and examined the trafficking of FasL during formation of immune synapses with HLA-deficient B cells., Results: FasL co-localized with CD63 more strongly than perforin or Lamp1+ in cytolytic granules. Electron microscopy revealed that FasL is enriched on intraluminal vesicles (ILVs) adjacent to the dense-core within cytolytic granules. In NK cells forming immune synapses with HLA-deficient B cells, a portion of FasL-containing granules re-localize toward the immune synapse, while a distinct pool of FasL remains at the distal pole of the cell., Conclusions: Localization of FasL to intra-luminal vesicles within cytolytic granules facilitates FasL trafficking to immune synapses and cytotoxic function in NK cells., (© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Egress of vaccinia virus from its host cell is mediated by the microtubule-associated motor kinesin-1, and three viral proteins, A36 and the F12/E2 complex, have been implicated in this process. Deletion of F12 expression causes a more severe reduction in egress than deletion of A36 but whether these proteins are involved in the same or different mechanisms of kinesin-1 recruitment is unknown. Here it is shown that a virus lacking both proteins forms a smaller plaque than mutants lacking either gene alone, indicating non-redundant functions. A36 not only links virions directly to kinesin-1 but also nucleates actin polymerization to propel surface virions away from the host cell. To address the relative importance of these functions for virus spread, a panel of recombinant viruses was constructed in which the ability of A36 to bind kinesin-1 or to nucleate actin polymerization was abrogated individually or together, in the presence or absence of F12 expression. Analysis of these viruses revealed that in the presence of the F12 protein, loss of kinesin-1 interaction made a greater contribution to plaque size than did the formation of actin tails. However in the absence of F12, the ability of A36 to promote egress was abrogated. Therefore, the ability of A36 to promote egress by kinesin-1 is reliant on the F12 protein.