A Bio-Liposome Activating Natural Killer Cell by Illuminating Tumor Homogenization Antigen Properties.

Natural killer (NK) cell therapies, primarily based on chimeric antigen receptor NK cells (CAR-NK), have been developed and applied clinically for therapeutic treatment of patients with mid-to-late-stage tumors. However, NK cell therapy has limited efficacy due to insufficient antigen expression on the tumor cell surface. Here, a universal "illuminate tumor homogenization antigen properties" (ITHAP) strategy to achieve stable and controlled antigen expression on the surface of tumor cells using nanomedicine, thus significantly enhancing the immune recognizability of tumor cells, is described. The ITHAP strategy is used to generate bio-liposomes (Pt@PL-IgG) composed of intermingled platelet membranes and liposomes with NK-activatable target antigen (IgG antibodies) and cisplatin pre-drug. It is demonstrated that Pt@PL-IgG successfully targets tumor cells using the autonomous drive of platelet membranes and achieves IgG implantation on tumor cells by utilizing membrane fusion properties. Moreover, it is shown that the Pt-DNA complex combined with NK cell-induced pyroptosis causes substantial interferon (IFN) secretion, thus providing a synthase-stimulator of interferon genes (STING)-IFN-mediated positive immune microenvironment to further potentiate NK therapy. These results show that anchoring cancer cells with NK-activatable target antigens is a promising translational strategy for addressing therapeutic challenges in tumor heterogeneity.

The role of monocytes and natural killers' immunophenotypic subsets in bronchial asthma in children.

Objective: Childhood bronchial asthma (BA) is a globally significant chronic disease with major health consequences. Recently, focus on the role of the innate immune system has been highlighted. Therefore, this study explores the role of circulating monocytes and natural killer (NK) clusters in childhood asthma.Methods: This case-control study enrolled 50 children with asthma divided equally into severe and mild groups and 26 healthy children. Flow-cytometry analysis was used to identify circulating blood monocytes and natural killers' subsets. In addition, pulmonary function test (spirometry) for children with asthma was performed.Results: This study showed significant negative correlations between frequency of total circulating, classical, intermediate, and nonclassical monocytes with ratio of forced expiratory volume/forced vital capacity (FEV1/FVC) (r = -0.637, P < 0.001; r = -0.575, P < 0.001; r = -0.657, P < 0.001; r = -0.329, P = 0.004, respectively). Also, there was significant negative correlations between frequency of total NKs and CD56dim CD16+ NK with FEV1/FVC (r = -0.584, P < 0.001) and (r = -0.579, P < 0.001). Significant predictors of childhood asthma severity were frequencies of total monocytes, total NKs, intermediate monocytes, and CD56dimCD16+ NK.Conclusion: Finally, we concluded that the FEV1/FVC is linked to aberrations of monocytes' and natural killers' immunophenotypic subsets in children with asthma. The frequencies of total monocytes and NK are significant predictors of severity of childhood asthma. The frequencies of CD14high CD16+ intermediate monocytes and CD56dim CD16+ NK cells are the best independent predictors of severity in children with asthma.

Multiomic analysis reveals cell-type-specific molecular determinants of COVID-19 severity.

The determinants of severe COVID-19 in healthy adults are poorly understood, which limits the opportunity for early intervention. We present a multiomic analysis using machine learning to characterize the genomic basis of COVID-19 severity. We use single-cell multiome profiling of human lungs to link genetic signals to cell-type-specific functions. We discover >1,000 risk genes across 19 cell types, which account for 77% of the SNP-based heritability for severe disease. Genetic risk is particularly focused within natural killer (NK) cells and T cells, placing the dysfunction of these cells upstream of severe disease. Mendelian randomization and single-cell profiling of human NK cells support the role of NK cells and further localize genetic risk to CD56bright NK cells, which are key cytokine producers during the innate immune response. Rare variant analysis confirms the enrichment of severe-disease-associated genetic variation within NK-cell risk genes. Our study provides insights into the pathogenesis of severe COVID-19 with potential therapeutic targets.

Synthetic DNA for Cell Surface Engineering: Experimental Comparison between Click Conjugation and Lipid Insertion in Terms of Cell Viability, Engineering Efficiency, and Displaying Stability.

The cell surface can be engineered with synthetic DNA for various applications ranging from cancer immunotherapy to tissue engineering. However, while elegant methods such as click conjugation and lipid insertion have been developed to engineer the cell surface with DNA, little effort has been made to systematically evaluate and compare these methods. Resultantly, it is often challenging to choose a right method for a certain application or to interpret data from different studies. In this study, we systematically evaluated click conjugation and lipid insertion in terms of cell viability, engineering efficiency, and displaying stability. Cells engineered with both methods can maintain high viability when the concentration of modified DNA is less than 25-50 µM. However, lipid insertion is faster and more efficient in displaying DNA on the cell surface than click conjugation. The efficiency of displaying DNA with lipid insertion is 10-40 times higher than that with click conjugation for a large range of DNA concentration. However, the half-life of physically inserted DNA on the cell surface is 3-4 times lower than that of covalently conjugated DNA, which depends on the working temperature. While the half-life of physically inserted DNA molecules on the cell surface is shorter than that of DNA molecules clicked onto the cell surface, lipid insertion is more effective than click conjugation in the promotion of cell-cell interactions under the two different experimental settings. The data acquired in this work are expected to act as a guideline for choosing an approximate method for engineering the cell surface with synthetic DNA or even other biomolecules.

The expression of intracellular cytokines of decidual natural killer cells in unexplained recurrent pregnancy loss.

OBJECTIVE: This study aims to investigate the expression levels of TNF-α, IFN-γ, IL-4, and IL-10 in dNK cells and determine whether or not the MAPK signal pathway is involved in the regulation of cytokine secretion by dNK cells at the maternal-fetal interface. METHODS: In this study, we collected decidua specimens from patients with apparently normal pregnant and unexplained recurrent pregnancy loss (URPL) and extracted dNK cells by enzymatic digestion. Then the expression of cytokines were analyzed by flow cytometry and Real-Time PCR respectively. RESULTS: The secretions of both IFN-γ and TNF-α in dNK cells in URPL were significantly higher than those in normal pregnancy. Furthermore, p38/MAPK inhibitors can inhibit the secretion of four cytokines in normal pregnancy, while in URPL cases, p38/MAPK inhibitors only significantly inhibit the secretion of IL-4 and IFN-γ. ERK inhibitors had no effect on the expression of all four cytokines and JNK/MAPK inhibitors varied on different cytokines. CONCLUSION: URPL is associated with a NK1 cytokine profile. MAPK signaling pathway is involved in the regulation of cytokine secretion by decidual NK cells at maternal-fetal interface.

Comprehensive Analyses of Type 1 Diabetes Ketosis- or Ketoacidosis-Related Genes in Activated CD56+CD16+ NK Cells.

Objectives: Alterations in natural killer (NK) cells activity cause damage to pancreatic islets in type 1 diabetes mellitus (T1DM). The aim of this study is to identify T1DM ketosis- or ketoacidosis-related genes in activated CD56+CD16+ NK cells. Methods: Microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were analyzed using the GEO2R tool. Enrichment analyses were performed using Metascape online database and GSEA software. Cell-specific gene co-expression network was built using NetworkAnalyst tools. Cytoscape software was used to identify hub genes and construct co-expressed networks. Target miRNAs were predicted based on the DIANA-micro T, miRDB, and miRWalk online databases. Results: A total of 70 DEGs were identified between T1DM patients recovered from ketosis or ketoacidosis and healthy control blood samples in GSE44314. Among the DEGs, 10 hub genes were screened out. The mature NK cell-specific gene co-expression network for DEGs in T1DM was built using NetworkAnalyst tools. DEGs between activated CD56+CD16+ NK cells and CD56brightCD16- NK cells were identified from GSE1511. After intersection, 13 overlapping genes between GSE44314 and GSE1511 microarray datasets were screened out, in which 7 hub genes were identified. Additionally, 59 target miRNAs were predicted according to the 7 hub genes. After validating with the exosome miRNA expression profile dataset of GSE97123, seven differentially expressed miRNAs (DEmiRNAs) in plasma-derived exosome were selected. Finally, a mRNA-miRNA network was constructed, which was involved in the T1DM ketosis or ketoacidosis process. Conclusion: This work identified seven hub genes in activated CD56+CD16+ NK cells and seven miRNAs in plasma-derived exosome as potential predictors of T1DM ketoacidosis, which provided a novel insight for the pathogenesis at the transcriptome level.

CAR-NK Cells: From Natural Basis to Design for Kill.

Chimeric antigen receptors (CARs) are fusion proteins with an extracellular antigen recognition domain and numerous intracellular signaling domains that have been genetically modified. CAR-engineered T lymphocyte-based therapies have shown great success against blood cancers; however, potential fatal toxicity, such as in cytokine release syndrome, and high costs are some shortcomings that limit the clinical application of CAR-engineered T lymphocytes and remain to overcome. Natural killer (NK) cells are the focal point of current immunological research owing to their receptors that prove to be promising immunotherapeutic candidates for treating cancer. However, to date, manipulation of NK cells to treat malignancies has been moderately successful. Recent progress in the biology of NK cell receptors has greatly transformed our understanding of how NK cells recognize and kill tumor and infected cells. CAR-NK cells may serve as an alternative candidate for retargeting cancer because of their unique recognition mechanisms, powerful cytotoxic effects especially on cancer cells in both CAR-dependent and CAR-independent manners and clinical safety. Moreover, NK cells can serve as an 'off-the-shelf product' because NK cells from allogeneic sources can also be used in immunotherapies owing to their reduced risk of alloreactivity. Although ongoing fundamental research is in the beginning stages, this review provides an overview of recent developments implemented to design CAR constructs to stimulate NK activation and manipulate NK receptors for improving the efficiency of immunotherapy against cancer, summarizes the preclinical and clinical advances of CAR-NK cells against both hematological malignancies and solid tumors and confronts current challenges and obstacles of their applications. In addition, this review provides insights into prospective novel approaches that further enhance the efficiency of CAR-NK therapies and highlights potential questions that require to be addressed in the future.

The Role of the HLA Class I α2 Helix in Determining Ligand Hierarchy for the Killer Cell Ig-like Receptor 3DL1.

HLA class I molecules that represent ligands for the inhibitory killer cell Ig-like receptor (KIR) 3DL1 found on NK cells are categorically defined as those HLA-A and HLA-B allotypes containing the Bw4 motif, yet KIR3DL1 demonstrates hierarchical recognition of these HLA-Bw4 ligands. To better understand the molecular basis underpinning differential KIR3DL1 recognition, the HLA-ABw4 family of allotypes were investigated. Transfected human 721.221 cells expressing HLA-A*32:01 strongly inhibited primary human KIR3DL1+ NK cells, whereas HLA-A*24:02 and HLA-A*23:01 displayed intermediate potency and HLA-A*25:01 failed to inhibit activation of KIR3DL1+ NK cells. Structural studies demonstrated that recognition of HLA-A*24:02 by KIR3DL1 used identical contacts as the potent HLA-B*57:01 ligand. Namely, the D1-D2 domains of KIR3DL1 were placed over the α1 helix and α2 helix of the HLA-A*24:02 binding cleft, respectively, whereas the D0 domain contacted the side of the HLA-A*24:02 molecule. Nevertheless, functional analyses showed KIR3DL1 recognition of HLA-A*24:02 was more sensitive to substitutions within the α2 helix of HLA-A*24:02, including residues Ile142 and Lys144 Furthermore, the presence of Thr149 in the α2 helix of HLA-A*25:01 abrogated KIR3DL1+ NK inhibition. Together, these data demonstrate a role for the HLA class I α2 helix in determining the hierarchy of KIR3DL1 ligands. Thus, recognition of HLA class I is dependent on a complex interplay between the peptide repertoire, polymorphisms within and proximal to the Bw4 motif, and the α2 helix. Collectively, the data furthers our understanding of KIR3DL1 ligands and will inform genetic association and immunogenetics studies examining the role of KIR3DL1 in disease settings.

Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells.

Many therapeutic monoclonal antibodies require binding to Fc γ receptors (FcγRs) for full effect and increasing the binding affinity increases efficacy. Preeminent among the five activating human FcγRs is FcγRIIIa/CD16a expressed by natural killer (NK) cells. CD16a is heavily processed, and recent reports indicate that the composition of the five CD16a asparagine(N)-linked carbohydrates (glycans) impacts affinity. These observations indicate that specific manipulation of CD16a N-glycan composition in CD16a-expressing effector cells including NK cells may improve treatment efficacy. However, it is unclear if modifying the expression of select genes that encode processing enzymes in CD16a-expressing effector cells is sufficient to affect N-glycan composition. We identified substantial processing differences using a glycoproteomics approach by comparing CD16a isolated from two NK cell lines, NK92 and YTS, with CD16a expressed by HEK293F cells and previous reports of CD16a from primary NK cells. Gene expression profiling by RNA-Seq and qRT-PCR revealed expression levels for glycan-modifying genes that correlated with CD16a glycan composition. These results identified a high degree of variability between the processing of the same human protein by different human cell types. N-glycan processing correlated with the expression of glycan-modifying genes and thus explained the substantial differences in CD16a processing by NK cells of different origins.

Microvesicles Produced by Natural Killer Cells Regulate the Formation of Blood Vessels.

We studied the effect of microvesicles derived from cells of the NK-92 cell line on the formation of tube-like structures by endothelial cells of the ЕА.Hy926 cell line. Microvesicles were isolated by differential centrifugation and their size was controlled by granulometric analysis using dynamic light scattering method. The effect of microvesicles produced by NK cells on angiogenesis was evaluated by cultural methods. In the course of the research, a model of co-culturing of microvesicles and endothelial cells on extracellular matrix Matrigel was developed. It was found that microvesicles derived from NK-92 cells promoted elongation of tube-like structures formed by endothelial ЕА.Hy926 cells. Microvesicles produced by NK cells can modulate functional activity of endothelial cells by affecting their ability to form blood vessels.

Effectual Labeling of Natural Killer Cells with Upconverting Nanoparticles by Electroporation for In Vivo Tracking and Biodistribution Assessment.

Natural killer (NK) cells, which are cytotoxic lymphocytes of the innate immune system and recognize cancer cells via various immune receptors, are promising agents in cell immunotherapy. To utilize NK cells as a therapeutic agent, their biodistribution and pharmacokinetics need to be evaluated following systemic administration. Therefore, in vivo imaging and tracking with efficient labeling and quantitative analysis of NK cells are required. However, the lack of the phagocytic capacity of NK cells makes it difficult to establish breakthroughs in cell labeling and subsequent in vivo studies. Herein, an effective labeling of upconverting nanoparticles (UCNPs) in NK cells is proposed using electroporation with high sensitivity and stability. The labeling performance of UCNPs functionalized with carboxy-polyethylene glycol (PEG) is better than with methoxy-PEG or with amine-PEG. The labeling efficiency becomes higher, but cell damage is greater as electric field increases; thus, there is an optimum electroporation condition for internalization of UCNPs into NK cells. The tracking and biodistribution imaging analyses of intravenously injected NK cells show that the labeled NK cells are initially distributed primarily in lungs and then spread to the liver and spleen. These advances will accelerate the application of NK cells as key components of immunotherapy against cancer.

Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells.

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

Elevated Cellular Oxidative Stress in Circulating Immune Cells in Otherwise Healthy Young People Who Use Electronic Cigarettes in a Cross-Sectional Single-Center Study: Implications for Future Cardiovascular Risk.

Background Tobacco cigarettes (TCs) increase oxidative stress and inflammation, both instigators of atherosclerotic cardiac disease. It is unknown if electronic cigarettes (ECs) also increase immune cell oxidative stress. We hypothesized an ordered, "dose-response" relationship, with tobacco-product type as "dose" (lowest in nonsmokers, intermediate in EC vapers, and highest in TC smokers), and the "response" being cellular oxidative stress (COS) in immune cell subtypes, in otherwise, healthy young people. Methods and Results Using flow cytometry and fluorescent probes, COS was determined in immune cell subtypes in 33 otherwise healthy young people: nonsmokers (n=12), EC vapers (n=12), and TC smokers (n=9). Study groups had similar baseline characteristics, including age, sex, race, and education level. A dose-response increase in proinflammatory monocytes and lymphocytes, and their COS content among the 3 study groups was found: lowest in nonsmokers, intermediate in EC vapers, and highest in TC smokers. These findings were most striking in CD14dimCD16+ and CD14++CD16+ proinflammatory monocytes and were reproduced with 2 independent fluorescent probes of COS. Conclusions These findings portend the development of premature cardiovascular disease in otherwise healthy young people who chronically vape ECs. On the other hand, that the COS is lower in EC vapers compared with TC smokers warrants additional investigation to determine if switching to ECs may form part of a harm-reduction strategy. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03823885.

Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles.

Natural killer (NK) cells form immune synapses to ascertain the state of health of cells they encounter. If a target cell triggers NK cell cytotoxicity, lytic granules containing proteins including perforin and granzyme B, are secreted into the synaptic cleft inducing target cell death. Secretion of these proteins also occurs from activated cytotoxic T lymphocytes (CTLs) where they have recently been reported to complex with thrombospondin-1 (TSP-1) in specialized structures termed supramolecular attack particles (SMAPs). Here, using an imaging method to define the position of each NK cell after removal, secretions from individual cells were assessed. NK cell synaptic secretion, triggered by ligation of NKp30 or NKG2D, included vesicles and SMAPs which contained TSP-1, perforin, and granzyme B. Individual NK cells secreted SMAPs, CD63+ vesicles, or both. A similar number of SMAPs were secreted per cell for both NK cells and CTLs, but NK cell SMAPs were larger. These data establish an unexpected diversity in NK cell synaptic secretions.

Favorable prognostic impact of Natural Killer cells and T cells in high-grade serous ovarian carcinoma.

Introduction: The aim of the present study was to investigate the prognostic impact of intratumoral cytotoxic T cells, Natural Killer (NK) cells, neutrophils and PD-L1 expression in patients with epithelial ovarian cancer.Methods: All patients diagnosed with high-grade serous carcinoma (HGSC) in Denmark in 2005 were included in the study. Immunohistochemical staining for PD-L1, CD8, CD66b and CD57 was performed on tumor tissue from 283 patients. Cell densities were analyzed using a digital image analysis method. The primary endpoint was overall survival (OS).Results: The median OS for HGSC patients was 30 months. It was 45 months in patients with high level of CD57+ NK cells (≥10 cells/mm2) compared with 29 month in patients with low level (<10 cells/mm2) (p = .0310). The median OS was 37 and 25 months in patients with high vs. low level of CD8+ T cells (cutoff 80 cells/mm2) (p = .0008). In multivariate analysis, high numbers of CD57+ NK cells and CD8+ T cells remained independent markers of favorable OS, adjusted hazard ratio (HR) 0.67; p = .041, and HR 0.72; p = .020, respectively. PD-L1 expression was associated with improved OS (37 months vs. 22 months, p = .0006), but was only borderline significant in the multivariate analysis (HR 0.77, p = .061). CD66b + neutrophils had no association with OS.Conclusions: In patients with HGSC tumor-infiltrating CD57+ NK cells and CD8+ T cells had favorable prognostic impact, while PD-L1 expression had borderline favorable prognostic significance. CD66b + neutrophils had no prognostic association. These findings may influence future immunotherapy development.

3D Nanochannel Electroporation for Macromolecular Nucleotide Delivery.

Delivery of macromolecular nucleotides into the living cells holds a great promise for the development of new therapeutics. However, its abilities for adoptive immunotherapy, cell reprogramming, and primary cell transfection have been long-term hindered by the lack of a system that can locally deliver engineered therapeutic nucleotides (e.g., plasmids, siRNAs, miRNAs) without causing any side effects. In this chapter, the performance of a novel 3D nanoelectroporation system (3D NEP) is highlighted in three scenarios-adoptive immunotherapy, cell reprogramming, and adult mouse primary cardiomyocyte transfection. Detailed protocols were given to introduce the 3D NEP system assembly, as well as their applications in (1) natural killer (NK) cells transfection by delivery of chimeric antigen receptor (CAR) plasmids; (2) mouse embryonic fibroblasts transfection with OSKM factors; and (3) miR-29b molecular beacon (BMs) delivery into primary cardiomyocytes for interrogating the side effect of miR-29b-assisted treatment.

VS38 Identifies Myeloma Cells With Dim CD38 Expression and Plasma Cells Following Daratumumab Therapy, Which Interferes With CD38 Detection for 4 to 6 Months.

OBJECTIVES: We report our institutional experience using VS38 to evaluate plasma cells by flow cytometry. METHODS: Flow cytometry data were reanalyzed to compare plasma cell percentages between the standard panel and VS38 panel. Natural killer (NK) and plasma cell CD38 median fluorescence intensity (MFI) values were calculated. RESULTS: Our cohort included 63 specimens from 38 patients. Twenty-six had received daratumumab (monoclonal anti-CD38 therapy) between less than 1 month and 17 months prior. For NK and plasma cells, CD38 MFI values were suppressed for 0 to 4 months and started to increase 4 to 6 months after last exposure. There was no significant difference in clonal plasma cell percentage calculated by the VS38 and standard panels; however, identification and quantification using the VS38 panel were easier. CONCLUSIONS: VS38 is a viable alternative to bright CD38 to identify plasma cells and particularly helpful in myeloma cases with dim CD38 and after daratumumab. Daratumumab interference with CD38 identification persists 4 to 6 months after the last exposure.

Optical Control of Cytokine Production Using Photoswitchable Galactosylceramides.

α-Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro-inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN-7000, the activity of which can be modulated with light. Based on established structure-activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro-inflammatory cytokines, such as IFN-γ. The azobenzene derivative α-GalACer-4 proved to be more potent than KRN-7000 itself when activated with 370 nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy.