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Chimeric antigen receptor (CAR) T cells have become an established immunotherapy with promising results for the treatment of hematological malignancies. However, modulation of the targeted antigen¡¯s surface level in cancer cells affects the quality and safety of CAR-T cell therapy. Here we present an engineered yeast-based antigen system for simulation of cancer cells with precise regulation of surface-antigen densities, providing a tool for controlled activation of CAR T cells and systematic assessment of antigen density effects. This Synthetic Cellular Advanced Signal Adapter (SCASA) system uses G protein-coupled receptor signaling to control cancer antigen densities on the yeast surface and provides a customizable platform allowing selectable signal inputs and modular pathway engineering for precise output fine-tuning. In relation to CD19+ cancers, we demonstrate synthetic cellular communication between CD19-displaying yeast and human CAR T cells as well as applications in high-throughput characterization of different CAR designs. We show that yeast is an alternative to conventional technologies (e.g. microbeads) and can provide higher activation control of clinically derived CAR T cells in vitro, relative to cancer cells. In summary, we present a customizable yeast-based platform for high-throughput characterization of CAR-T cell functionality and show potential applications within therapeutic T cells in clinical settings. Chimeric antigen receptor T (CAR-T) cell therapy uses engineered donor T cells to recognize and eliminate cancer cells through cognate antigen-dependent activation. Here authors develop an alternative to bead-based or cancer-cell-induced CAR-T cell activation by presenting the antigen on the surface of engineered yeast cells, which allows precise regulation of antigen density.

Enhancers and transcription factors (TFs) are crucial in regulating cellular processes. Current multiomic technologies to study these elements in gene regulatory mechanisms lack multiplexing capability and scalability. Here we present single-cell ultra-high-throughput multiplexed sequencing (SUM-seq) for co-assaying chromatin accessibility and gene expression in single nuclei. SUM-seq enables profiling hundreds of samples at the million cell scale and outperforms current high-throughput single-cell methods. We demonstrate the capability of SUM-seq to (1) resolve temporal gene regulation of macrophage M1 and M2 polarization to bridge TF regulatory networks and immune disease genetic variants, (2) define the regulatory landscape of primary T helper cell subsets and (3) dissect the effect of perturbing lineage TFs via arrayed CRISPR screens in spontaneously differentiating human induced pluripotent stem cells. SUM-seq offers a cost-effective, scalable solution for ultra-high-throughput single-cell multiomic sequencing, accelerating the unraveling of complex gene regulatory networks in cell differentiation, responses to perturbations and disease studies. This work presents SUM-seq, an ultra-high-throughput method for co-profiling chromatin accessibility and gene expression in single nuclei across multiplexed samples, advancing the study of gene regulation in diverse biological systems.

SummaryHemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood, is associated with infections by Escherichia coli strains producing Shiga toxin 2 (Stx2). The microangiopathic injuries caused by the toxin occur mainly in the renal microvasculature when the glycolipid receptor globotriaosylceramide (Gb3Cer) is targeted. Before entering the kidney, Stx2 binds to circulating cells through Gb3Cer and Toll-like receptor 4 (TLR4) and is subsequently delivered in extracellular vesicles to target cells. Here, we have found a specific inhibitor of the Stx2/TLR4 interaction, the preclinical polymyxin B derivative NAB815. The compound impairs the formation of Stx2-containing extracellular vesicles produced by leukocytes and platelets and also reduces their toxic effects in cellular (Vero cells) and animal models (CD-1 mice). NAB815 would represent a useful tool in preventing HUS and is effective at sub-bactericidal concentrations, thus overcoming the concern that antibiotics are harmful to patients infected with Stx2-producing E. coli. Graphical abstract Highlights?In children, life-threatening HUS is caused by bacteria producing Shiga toxins?There are no specific treatments for HUS, and antibiotics are not recommended?We have found an antibiotic impairing toxin actions at sub-bactericidal concentration?Administration of this drug in patients may represent a useful tool in preventing HUS Pharmacology; Natural sciences; Biological sciences; Microbiology; Medical Microbiology