New Insight into Intestinal Mast Cells Revealed by Single-Cell RNA Sequencing.

Mast cells (MCs) are tissue-resident immune cells distributed in all tissues and strategically located close to blood and lymphatic vessels and nerves. Thanks to the expression of a wide array of receptors, MCs act as tissue sentinels, able to detect the presence of bacteria and parasites and to respond to different environmental stimuli. MCs originate from bone marrow (BM) progenitors that enter the circulation and mature in peripheral organs under the influence of microenvironment factors, thus differentiating into heterogeneous tissue-specific subsets. Even though MC activation has been traditionally linked to IgE-mediated allergic reactions, a role for these cells in other pathological conditions including tumor progression has recently emerged. However, several aspects of MC biology remain to be clarified. The advent of single-cell RNA sequencing platforms has provided the opportunity to understand MCs' origin and differentiation as well as their phenotype and functions within different tissues, including the gut. This review recapitulates how single-cell transcriptomic studies provided insight into MC development as well as into the functional role of intestinal MC subsets in health and disease.

Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles.

The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.