UNASSIGNED: Age-related Macular Degeneration (AMD) poses a growing global health concern as the leading cause of central vision loss in elderly people.
UNASSIGNED: This study focuses on unraveling the intricate involvement of Natural Killer (NK) cells in AMD, shedding light on their immune responses and cytokine regulatory roles.
UNASSIGNED: Transcriptomic data from the Gene Expression Omnibus database were utilized, employing single-cell RNA-seq analysis. High-dimensional weighted gene co-expression network analysis (hdWGCNA) and single-cell regulatory network inference and clustering (SCENIC) analysis were applied to reveal the regulatory mechanisms of NK cells in early-stage AMD patients. Machine learning models, such as random forests and decision trees, were employed to screen hub genes and key transcription factors (TFs) associated with AMD.
UNASSIGNED: Distinct cell clusters were identified in the present study, especially the T/NK cluster, with a notable increase in NK cell abundance observed in AMD. Cell-cell communication analyses revealed altered interactions, particularly in NK cells, indicating their potential role in AMD pathogenesis. HdWGCNA highlighted the turquoise module, enriched in inflammation-related pathways, as significantly associated with AMD in NK cells. The SCENIC analysis identified key TFs in NK cell regulatory networks. The integration of hub genes and TFs identified CREM, FOXP1, IRF1, NFKB2, and USF2 as potential predictors for AMD through machine learning.
UNASSIGNED: This comprehensive approach enhances our understanding of NK cell dynamics, signaling alterations, and potential predictive models for AMD. The identified TFs provide new avenues for molecular interventions and highlight the intricate relationship between NK cells and AMD pathogenesis. Overall, this study contributes valuable insights for advancing our understanding and management of AMD.

UNASSIGNED: The immunological characteristics that could protect children with coronavirus disease 2019 (COVID-19) from severe or fatal illnesses have not been fully understood yet.
UNASSIGNED: Here, we performed single-cell RNA sequencing (scRNA-seq) analysis on peripheral blood samples of 15 children (8 with COVID-19) and compared them to 18 adults (13 with COVID-19).
UNASSIGNED: The child-adult integrated single cell data indicated that children with the disease presented a restrained response to type I interferon in most of the major immune cell types, along with suppression of upstream interferon regulatory factor and toll-like receptor expression in monocytes, which was confirmed by in vitro interferon stimulation assays. Unlike adult patients, children with COVID-19 showed lower frequencies of activated proinflammatory CD14+ monocytes, possibly explaining the rareness of cytokine storm in them. Notably, natural killer (NK) cells in pediatric patients displayed potent cytotoxicity with a rich expression of cytotoxic molecules and upregulated cytotoxic pathways, whereas the cellular senescence, along with the Notch signaling pathway, was significantly downregulated in NK cells, all suggesting more robust cytotoxicity in NK cells of children than adult patients that was further confirmed by CD107a degranulation assays. Lastly, a modest adaptive immune response was evident with more naïve T cells but less activated and proliferated T cells while less naïve B cells but more activated B cells in children over adult patients.
UNASSIGNED: Conclusively, this preliminary study revealed distinct cell frequency and activation status of major immune cell types, particularly more robust NK cell cytotoxicity in PBMC that might help protect children from severe COVID-19.

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disease caused by mutations in effectors and regulators of cytotoxicity in cytotoxic T cells (CTL) and natural killer (NK) cells. The complexity of the immune system means that in vivo models are needed to efficiently study diseases like HLH. Mice with defects in the genes known to cause primary HLH (pHLH) are available. However, these mice only develop the characteristic features of HLH after the induction of an immune response (typically through infection with lymphocytic choriomeningitis virus). Nevertheless, murine models have been invaluable for understanding the mechanisms that lead to HLH. For example, the cytotoxic machinery (e.g., the transport of cytotoxic vesicles and the release of granzymes and perforin after membrane fusion) was first characterized in the mouse. Experiments in murine models of pHLH have emphasized the importance of cytotoxic cells, antigen-presenting cells (APC), and cytokines in hyperinflammatory positive feedback loops (e.g., cytokine storms). This knowledge has facilitated the development of treatments for human HLH, some of which are now being tested in the clinic.

The laboratory diagnosis of cytokine storm syndromes (CSSs), i.e., hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS), is often challenging. The laboratory features using routinely available tests lack specificity, whereas confirmatory testing is available in only few laboratories in the United States. The disease mechanisms are still largely unclear, particularly in adults. In this chapter, the pathogenesis of CSSs, their associated laboratory findings, and recommended diagnostic strategies are reviewed.

Production of large amounts of functional NK and CAR-NK cells represents one of the bottlenecks for NK-based immunotherapy. In this study, we developed a large-scale, reliable, and practicable NK and CAR-NK production using G-Rex 100M bioreactors, which depend on a gas-permeable membrane technology. This system holds large volumes of medium with enhanced oxygen delivery, creating conditions conducive to large-scale PBNK and CAR-NK expansions for cancer therapy. Both peripheral blood NK cells (PBNKs) and CAR-NKs expanded in these bioreactors retained similar immunophenotypes and exhibited comparable cytotoxicity towards hepatocellular carcinoma (HCC) cells akin to that of NK and CAR-NK cells expanded in G-Rex 6 well bioreactors. Importantly, cryopreservation minimally affected the cytotoxicity of NK cells expanded using the G-Rex 100M bioreactors, establishing a robust platform for scaled-up NK and CAR-NK cell production. This method is promising for the development of \"off-the-shelf\" NK cells, supporting the future clinical implementation of NK cell immunotherapy.

NK cells participate in ischemia reperfusion injury (IRI) and transplant rejection. Endogenous regulatory systems may exist to attenuate NK cell activation and cytotoxicity in IRI associated with kidney transplantation. A greater understanding of NK regulation will provide insights in transplant outcomes and could direct new therapeutic strategies. Kidney tubular epithelial cells (TECs) may negatively regulate NK cell activation by their surface expression of a complex family of C-type lectin-related proteins (Clrs). We have found that Clr-b and Clr-f were expressed by TECs. Clr-b was upregulated by inflammatory cytokines TNFα and IFNγ in vitro. Silencing of both Clr-b and Clr-f expression using siRNA resulted in increased NK cell killing of TECs compared to silencing of either Clr-b or Clr-f alone (p < 0.01) and when compared to control TECs (p < 0.001). NK cells treated in vitro with soluble Clr-b and Clr-f proteins reduced their capacity to kill TECs (p < 0.05). Hence, NK cell cytotoxicity can be inhibited by Clr proteins on the surface of TECs. Our study suggests a synergistic effect of Clr molecules in regulating NK cell function in renal cells and this may represent an important endogenous regulatory system to limit NK cell-mediated organ injury during inflammation.

UNASSIGNED: Natural killer (NK) cell-based therapies are a promising new method for treating indolent cancer, however engineering new therapies is complex and progress towards therapy for solid tumors is slow. New methods for determining the underlying intracellular signaling driving the killing phenotype would significantly improve this progress.
UNASSIGNED: We combined single-cell RNA sequencing with live cell imaging of a model system of NK cell killing to correlate transcriptomic data with functional output. A model of NK cell activity, the NK-92 cell line killing of HeLa cervical cancer cells, was used for these studies. NK cell killing activity was observed by microscopy during co-culture with target HeLa cells and killing activity subsequently manually mapped based on NK cell location and Annexin V expression. NK cells from this culture system were profiled by single-cell RNA sequencing using the 10× Genomics platform, and transcription factor activity inferred using the Viper and DoRothEA R packages. Luminescent microscopy of reporter constructs in the NK cells was then used to correlate activity of inferred transcriptional activity with killing activity.
UNASSIGNED: NK cells had heterogeneous killing activity during 10 h of culture with target HeLa cells. Analysis of the single cell sequencing data identified Nuclear Factor Kappa B (NF-κB), Signal Transducer and Activator of Transcription 1 (STAT1) and MYC activity as potential drivers of NK cell functional phenotype in our model system. Live cell imaging of the transcription factor activity found NF-κB activity was significantly correlated with past killing activity. No correlation was observed between STAT1 or MYC activity and NK cell killing.
UNASSIGNED: Combining luminescent microscopy of transcription factor activity with single-cell RNA sequencing is an effective means of assigning functional phenotypes to inferred transcriptomics data.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s12195-024-00812-3.

BACKGROUND: For decades, immunotherapies have been integral for the treatment and management of bladder cancer, with immune checkpoint inhibitors (ICIs) transforming patient care in recent years. However, response rates are poor to T cell-targeted ICIs such as programmed cell death protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) blocking antibodies, framing a critical need for complementary immunotherapies. Promising strategies involve harnessing the activation potential of natural killer (NK) cells. They quickly exert their antitumor activity via signaling through germline-encoded activating receptors and are rapidly sensitized to new tissue microenvironments via their regulation by polymorphic HLA class I, KIR and NKG2A receptors.
OBJECTIVE: In this review, we examined the roles of currently available NK-targeted antitumor treatment strategies such as engineered viral vectors, small-molecule IMiDs, NK agonist antibodies, interleukins, and chimeric antigen receptor (CAR) NK cells, and their potential for improving the efficacy of immunotherapy in the treatment of bladder cancer.
METHODS: Through review of current literature, we summarized our knowledge of NK cells in solid tumors and hematologic malignancies as their roles pertain to novel immunotherapies already being applied to the treatment of bladder cancer or that offer rationale for considering as potential novel immunotherapeutic strategies.
RESULTS: NK cells play a critical role in shaping the tumor microenvironment (TME) that can be exploited to improve T cell-targeted immunotherapies.
CONCLUSIONS: Emerging evidence suggests that NK cells are a prime target for improving antitumor functions in immunotherapies for the treatment of bladder cancer. Further research into profiling NK cells in settings of immunotherapies for bladder cancer could help identify patients who might maximally benefit from NK cell-targeted immunotherapies and the various approaches for exploiting their antitumor properties.

UNASSIGNED: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder characterized by uncontrolled lymphocyte and macrophage activation and a subsequent cytokine storm. The timely initiation of immunosuppressive treatment is crucial for survival.
UNASSIGNED: Here, we harnessed Vγ9Vδ2 T cell degranulation to develop a novel functional assay for the diagnosis of HLH. We compared the novel assay with the conventional natural killer (NK) cell stimulation method in terms of efficiency, specificity, and reliability. Our analysis involved 213 samples from 182 individuals, including 23 samples from 12 patients with degranulation deficiency (10 individuals with UNC13D deficiency, 1 with STXBP2 deficiency, and 1 with RAB27A deficiency).
UNASSIGNED: While both tests exhibited 100% sensitivity, the Vγ9Vδ2 T cell degranulation assay showed a superior specificity of 86.2% (n=70) compared to the NK cell degranulation assay, which achieved 78.9% specificity (n=213). The Vγ9Vδ2 T cell degranulation assay offered simpler technical requirements and reduced labor intensity, leading to decreased susceptibility to errors with faster processing times.
UNASSIGNED: This efficiency stemmed from the sole requirement of dissolving (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) powder, contrasting with the intricate maintenance of K562 cells necessary for the NK cell degranulation assay. With its diminished susceptibility to errors, we anticipate that the assay will require fewer repetitions of analysis, rendering it particularly well-suited for testing infants.
UNASSIGNED: The Vγ9Vδ2 T cell degranulation assay is a user-friendly, efficient diagnostic tool for HLH. It offers greater specificity, reliability, and practicality than established methods. We believe that our present findings will facilitate the prompt, accurate diagnosis of HLH and thus enable rapid treatment and better patient outcomes.

UNASSIGNED: This study investigated potential predictive models associated with natural killer (NK) cell mitochondrial membrane potential (MMP or ΔΨm) in predicting death among critically ill patients with COVID-19.
UNASSIGNED: We included 97 patients with COVID-19 of different severities attending Peking Union Medical College Hospital from December 2022 to January 2023. Patients were divided into three groups according to oxygen and mechanical ventilation use during specimen collection and were followed for survival and death at 3 months. The lymphocyte subpopulation MMP was detected via flow cytometry. We constructed a joint diagnostic model by integrating identified key indicators and generating receiver operating curves (ROCs) and evaluated its predictive performance for mortality risk in critically ill patients.
UNASSIGNED: The NK-cell MMP median fluorescence intensity (MFI) was significantly lower in critically ill patients who died from COVID-19 (p<0.0001) and significantly and positively correlated with D-dimer content in critically ill patients (r=0.56, p=0.0023). The random forest model suggested that fibrinogen levels and NK-cell MMP MFI were the most important indicators. Integrating the above predictive models for the ROC yielded an area under the curve of 0.94.
UNASSIGNED: This study revealed the potential of combining NK-cell MMP with key clinical indicators (D-dimer and fibrinogen levels) to predict death among critically ill patients with COVID-19, which may help in early risk stratification of critically ill patients and improve patient care and clinical outcomes.