Bottom-up assembly of target-specific cytotoxic synthetic cells.

Immune vigilance ensures body integrity by eliminating malignant cells through the complex but coordinated cooperation of highly diversified lymphocytes populations. The sheer complexity of the immune system has slowed development of immunotherapies based on top-down genetic engineering of lymphocytes. In contrast, bottom-up assembly of synthetic cell compartments has contributed novel engineering strategies to reverse engineer and understand cellular phenomena as molecularly defined systems. Towards reducing the complexity of immunological systems, herein, a bottom-up approach for controlled assembly of fully-synthetic immune-inspired cells from predefined molecular components based on giant unilamellar vesicles is described. For construction of target-specific cytotoxic immune cells, the Fas-ligand-based apoptosis-inducing immune cell module is combined with an antibody-mediated cellular cytotoxicity-inspired system. The designed immune cells identify leukemia cells by specific surface antigens. Subsequently, they form stable attachments sites and eliminate their targets by induction of apoptosis. A structural and functional characterization of the synthetic immune cells by means of microfluidics, live cell, confocal and electron microscopy, dynamic light scattering as well as flow cytometry is presented. This study demonstrates the bioinspired construction of effector immune cells from defined molecular building blocks, enabling learning-by-building approaches in synthetic immunology.

Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle-Based Protein Signaling.

Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes, especially those involved in immune signalling. However, the mechanistic understanding concerning the biophysical principles underlying EV-based communication is still incomplete. Towards holistic understanding, particular mechanisms explaining why and when cells apply EV-based communication and how protein-based signalling is promoted by EV surfaces are sought. Here, the authors study vesicle-induced receptor sequestration (VIRS) as a universal mechanism augmenting the signalling potency of proteins presented on EV-membranes. By bottom-up reconstitution of synthetic EVs, the authors show that immobilization of the receptor ligands FasL and RANK on EV-like vesicles, increases their signalling potential by more than 100-fold compared to their soluble forms. Moreover, the authors perform diffusion simulations within immunological synapses to compare receptor activation between soluble and EV-presented proteins. By this the authors propose vesicle-triggered local clustering of membrane receptors as the principle structural mechanism underlying EV-based protein presentation. The authors conclude that EVs act as extracellular templates promoting the local aggregation of membrane receptors at the EV contact site, thereby fostering inter-protein interactions. The results uncover a potentially universal mechanism explaining the unique structural profit of EV-based intercellular signalling.

Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.