Although great advances in siRNA and mRNA delivery have been made (e.g. with lipid nanoparticles), there remains a pressing need for a versatile platform that can deliver both RNA and protein payloads with convenient processing and storage. Here, we demonstrate a highly efficient approach to forming ca. 100 nm vesicular polymer nanoparticles (polymersomes) that requires no organic solvents by utilizing thermoresponsive block copolymers capable of self-assembling in aqueous medium as the solution is raised to room temperature. This is achieved using block copolymers with a domain that has a lower critical solution temperature (LCST) such that they are soluble in aqueous medium under standard refrigeration (4-7°C) temperature but assemble upon warming to room temperature, resulting in large batches of nanoparticles with predictable size and morphology as dictated by polymer structure. The nanomaterials are designed with charged and biofunctional moieties to drive payload affinity as well as in-vivo targeting, respectively. Both siRNA and protein payloads can be incorporated during warming at higher than 75% loading efficiencies. Payload-polymer suspensions can also be lyophilized into a dry state, allowing for greater hydrolytic stability under mild (4-7°C) refrigeration conditions. This greatly reduces processing and storage requirements, as the powder can simply be reconstituted in cold aqueous medium, then used as described. Here, we selected biomedical applications that did not require removal of unencapsulated payload, bypassing further purification processes and demonstrating a highly scalable approach for formulating nanoparticle-based treatments. Finally, we demonstrate that our system is capable of in-vivo delivery in the contexts of protein subunit vaccination, prophylactic immune tolerance induction, and siRNA interference therapy in cancer. We believe this polymersome platform addresses several challenges to broad translation of nanoparticle delivery of biologics to the clinic, such as scalability, loading efficiency, quality control, and dry storage.

The therapeutic potential for human type 2 innate lymphoid cells (ILC2s) has been underexplored. Although not observed in mouse ILC2s, we found that human ILC2s secrete granzyme B (GZMB) and directly lyse tumor cells by inducing pyroptosis and/or apoptosis, which is governed by a DNAM-1-CD112/CD155 interaction that inactivates the negative regulator FOXO1. Over time, the high surface density expression of CD155 in acute myeloid leukemia cells impairs the expression of DNAM-1 and GZMB, thus allowing for immune evasion. We describe a reliable platform capable of up to 2,000-fold expansion of human ILC2s within 4 weeks, whose molecular and cellular ILC2 profiles were validated by single-cell RNA sequencing. In both leukemia and solid tumor models, exogenously administered expanded human ILC2s show significant antitumor effects in vivo. Collectively, we demonstrate previously unreported properties of human ILC2s and identify this innate immune cell subset as a member of the cytolytic immune effector cell family.
Copyright © 2023 Elsevier Inc. All rights reserved.

The mechanisms that guide Th2 cell differentiation in barrier tissues are unclear. Using temporal, spatial and single cell transcriptomic tracking of house dust mite (HDM) specific T cells, we describe the molecular pathways driving allergen specific Th2 cells. Differentiation and migration of lung allergen-specific Th2 cells requires early expression of the transcriptional repressor Blimp-1. Loss of Blimp-1 during priming in the lymph node ablated the formation of Th2 cells that migrate to the lung, indicating early Blimp-1 promotes the population of Th2 cells with migratory capability. Blimp-1 occurs in a subset of lymph node CD4 T cells that requires IL-10 from allergen-specific T cells. Furthermore, IL-2/STAT5 signals are essential for both Blimp-1 and GATA3 upregulation through repression of Bcl6 and Bach2 in the lymph node. Spatial microniches of IL-2 in the lymph node identified by the latent factor discovery method SLIDE discriminate and support the earliest Blimp-1+ migratory Th2 cells, demonstrating that lymph node localization is a primary driver of Th2 initiation. Our findings illuminate the molecular pathways for inhaled allergens to promote Th2 cells and identify an early requirement for IL-2 mediated spatial microniches that synergize with allergen-driven IL-10 from responding T cells to drive allergic asthma

Knowledge of the transcriptional programs underpinning the functions of human kidney cell populations at homeostasis is limited. We present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 cells to map human kidney at high resolution. Sex-based differences in gene expression within proximal tubular cells were observed, specifically, increased anti-oxidant metallothionein genes in females and aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed in proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. We identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions. Significant heterogeneity in myeloid cells was observed, with a MRC1+LYVE1+FOLR2+C1QC+ population representing a predominant population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, and explores the complexity of parenchymal and kidney-resident immune cells.
© 2022. The Author(s).

Maintaining organ homeostasis requires complex functional synergy between distinct cell types, a snapshot of which is glimpsed through the simultaneously broad and granular analysis provided by single-cell atlases. Knowledge of the transcriptional programs underpinning the complex and specialized functions of human kidney cell populations at homeostasis is limited by difficulty accessing healthy, fresh tissue. Here, we present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 high-quality cells to map healthy kidney at high resolution. Our sex-balanced dataset revealed sex-based differences in gene expression within proximal tubular cells, specifically, increased anti-oxidant metallothionein genes in females and the predominance of aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed between male and female proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. Within the immune niche, we identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions and validated findings by flow cytometry. We observed significant heterogeneity in resident myeloid populations and identified an MRC1 + LYVE1 + FOLR2 + C1QC + population as the predominant myeloid population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, revealing novel insights into the complexity of renal parenchymal cells and kidney-resident immune populations.