Natural killer (NK) cells are potent cytotoxic innate lymphocytes that can be used for cancer immunotherapy. Since the balance of signals from activating and inhibitory receptors determines the activity of NK cells, their anti-tumor activity can be potentiated by overexpressing activating receptors or knocking out inhibitory receptors via genome engineering, such as chimeric antigen receptor (CAR) transgenesis and CRISPR-Cas9-mediated gene editing, respectively. Here, we report the development of a one-step strategy for CRISPR-Cas9-mediated gene knockout and CAR transgenesis in NK cells using retroviral particles. We generated NK cells expressing anti-epidermal growth factor receptor (EGFR)-CAR with simultaneous TIGIT gene knockout using single transduction and evaluated the consequence of the genetic modifications in vitro and in vivo. Taken together, our results demonstrate that retroviral particle-mediated engineering provides a strategy readily applicable to simultaneous genetic modifications of NK cells for efficient immunotherapy.
Copyright © 2023.

Chlamydia pneumoniae (Cpn) is a gram-negative intracellular pathogen that causes a variety of pulmonary diseases, and there is growing evidence that it may play a role in Alzheimer's disease (AD) pathogenesis. Cpn can interact functionally with host histones, altering the host's epigenetic regulatory system by introducing bacterial products into the host tissue and inducing a persistent inflammatory response. Because Cpn is difficult to propagate, isolate, and detect, a modified LPS-like neuroinflammation model was established using lyophilized cell free supernatant (CFS) obtained from infected cell cultures, and the effects of CFS were compared to LPS. The neuroprotective effects of Trichostatin A (TSA), givinostat, and RG108, which are effective on epigenetic mechanisms, and the antibiotic rifampin, were studied in this newly introduced model and in the presence of amyloid beta (Aβ) 1-42. The neuroprotective effects of the drugs, as well as the effects of CFS and LPS, were evaluated in Aβ-induced neurotoxicity using a real-time cell analysis system, total ROS, and apoptotic impact. TSA, RG108, givinostat, and rifampin all demonstrated neuroprotective effects in both this novel model and Aβ-induced neurotoxicity. The findings are expected to provide early evidence on neuroprotective actions against Cpn-induced neuroinflammation and Aβ-induced neurotoxicity, which could represent a new treatment option for AD, for which there are currently few treatment options.

High-throughput single-cell RNA sequencing (scRNA-seq) has become a frequently used tool to assess immune cell heterogeneity. Recently, the combined measurement of RNA and protein expression was developed, commonly known as cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcripts but also nearly doubles the sequencing read depth required per single cell. Furthermore, there is still a paucity of analysis tools to visualize combined transcript-protein datasets. Here, we describe a targeted transcriptomics approach that combines an analysis of over 400 genes with simultaneous measurement of over 40 proteins on 2 × 104 cells in a single experiment. This targeted approach requires only about one-tenth of the read depth compared to a whole-transcriptome approach while retaining high sensitivity for low abundance transcripts. To analyze these multi-omic datasets, we adapted one-dimensional soli expression by nonlinear stochastic embedding (One-SENSE) for intuitive visualization of protein-transcript relationships on a single-cell level.
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

Soybeans are a rich source of isoflavones that have been linked with anti-inflammatory processes and various health benefits. However, specific mechanisms whereby soy bioactives impact immune cell subsets are unclear. Isoflavones, such as genistein and daidzein, are metabolized by microbes to bioactive metabolites as O-desmethylangolensin (O-DMA) and equol, whose presence has been linked to health benefits. We examined how soy isoflavones and metabolites impact natural killer (NK) cell signaling and function. We observe no impact of isoflavones on viability of healthy donor peripheral blood mononuclear cells (PBMCs) or NK cells, even at high (25 µM) concentrations. However, pre-treatment of PBMCs with physiologically-relevant concentrations of genistein (p = 0.0023) and equol (p = 0.006) decreases interleukin (IL)-12/IL-18-induced interferon-gamma (IFN-γ) production versus controls. Detailed cellular analyses indicate genistein and equol decrease IL-12/IL-18-induced IFN-γ production by human NK cell subsets, but do not consistently alter cytotoxicity. At the level of signal transduction, genistein decreases IL-12/IL-18-induced total phosphorylated tyrosine, and phosphorylation MAPK pathway components. Further, genistein limits IL-12/IL-18-mediated upregulation of IL-18Rα expression on NK cells (p = 0.0109). Finally, in vivo studies revealed that C57BL/6 mice fed a soy-enriched diet produce less plasma IFN-γ following administration of IL-12/IL-18 versus control-fed animals (p < 0.0001). This study provides insight into how dietary soy modulates NK cell functions.