Regulatory roles of transcription factors T-bet and Eomes in group 1 ILCs.

T-bet and Eomes, both T-box transcription factors, have been extensively studied for their critical roles in the differentiation and functional maintenance of various immune cells. In this review, we provide a focused overview of their contributions to the transcriptional activation and differentiation, development, and terminal maturation of natural killer cells and innate lymphoid cell 1 cells. Furthermore, the interplay between T-bet and Eomes in regulating NK cell function, and its subsequent implications for immune responses against infections and tumors, is thoroughly examined. The review explores the ramifications of dysregulated transcription factor expression, examining its impact on homeostatic balance and its role in a spectrum of disease models. Expression variances among distinct NK cell subsets resident in different tissues are highlighted to underscore the complexity of their biological roles. Collectively, this work aims to expand the current understanding of NK cell biology, thereby paving the way for innovative approaches in the realm of NK cell-based immunotherapies.

Human Leukocyte Antigen Class I Expression and Natural Killer Cell Infiltration and Its Correlation with Prognostic Features in Luminal Breast Cancers.

Purpose: The aim of this study was to determine whether low HLA-I expression and NK cells infiltration are related to prognostic features in breast cancer, as observed in cancers in other locations and non-hormone dependent breast cancers. Particularly, we explored their relation to infiltrated axillary lymph nodes (ALNs), with the aim of finding new predictors helping to decide the extent of axillary surgery. Patients and Methods: We conducted a retrospective correlational analysis of 35 breast cancers from 35 breast cancer patients showing axillary infiltration at diagnosis and with upfront surgery. HLA-I H-score and the number of NK cells x 50 high power fields (HPF) in the biopsy specimen were correlated with pathological variables of the surgical specimen: number of infiltrated ALNs, tumor size, histological type, the presence of ductal carcinoma in situ, focality, histological grade, necrosis, lymphovascular and perineural invasion, Her2Neu status, and the percentages of tumor-infiltrating lymphocytes (TILs), estrogen receptor, progesterone receptor, ki67, and p53. Results: All tumors showed hormone receptor expression and three of them Her2Neu positivity. A positive correlation (p=0.001**) was found between HLA-I H-score and TILs and Ki67 expression. HLA H-score increased with histological grade and was higher in unifocal than in multifocal disease (p=0.044 and p=0.011, respectively). No other correlations were found. Conclusion: High HLA-I H-score values correlated with features of poor prognosis in this cohort of luminal breast tumors, but not with infiltrated ALNs. This finding highlights the differences between luminal breast cancer, and cancers in other locations and non-hormone dependent breast cancers, in which low HLA-I expression tends to be associated with poor prognostic features.

miR-17-5p/STAT3/H19: A novel regulatory axis tuning ULBP2 expression in young breast cancer patients.

BACKGROUND AND AIM: UL-16 binding protein 2 (ULBP2) is a highly altered ligand for the activating receptor, NKG2D in breast cancer (BC). However, the mechanism behind its de-regulation in BC patients remains to be explored. The sophisticated crosstalk between miR-17-5p, the lncRNA H19, and STAT3 as a possible upstream regulatory loop for ULBP2 in young BC patients and cell lines remains as an unexplored area. Therefore, this study aimed at unravelling the ncRNA circuit regulating ULBP2 in young BC patients and cell lines. PATIENTS AND METHODS: A total of 30 BC patients were recruited for this study. The expression levels of miR-17-5p, lncRNA H19, and STAT3 were examined in 30 BC tissues compared to their normal counterparts. In addition, the expression signatures of those transcripts were compared in young (<40 years) and old BC (≥40 years) patients. miR-17-5p oligonucleotides, STAT3 and H19 siRNAs were transfected in MDA-MB-231 cells using HiPerfect® Transfection Reagent. miR-17-5p and the transcripts of the target genes quantified using RT-qPCR. Their relative expression was calculated using the 2-ΔΔCT method. RESULTS: Through acting as a ceRNA circuit that antagonizes the function of miR-17-5p, H19 prevented the miR-17-5p-induced downregulation of STAT3; this mechanism further contributes to the pathogenesis of BC. Ectopic expression of miR-17-5p in MDA-MB-231 cells displayed its prominent role as an indirect potential activator of NK cells by significantly repressing the expression levels of the oncogenic mediator STAT3 and the oncogenic lncRNA H19 and inducing ULBP2 expression level by 3 folds in TNBC cell lines compared to mock cells. Furthermore, knocking down of STAT3 repressed the lncRNA H19 and increased ULBP2 expression levels, whereas siRNAs against H19 increased the expression levels of ULBP2. CONCLUSION: This study highlighted the crosstalk between the novel regulatory network composed of miR-17-5p, H19 and STAT3, and their impact on ULBP2 in BC. Moreover, this study underscored the potential role of miR-17-5p in counteracting the immune evasion tactics, particularly the shedding of ULBP2 in young BC patients, through the modulation of the STAT3/H19/ULBP2 regulatory axis. Thus, targeting this novel regulatory network could potentially enhance our understanding and advance the future application of the innate system-mediated immunotherapy in BC.

Immunomodulatory effects of immune cell-derived extracellular vesicles in melanoma.

Melanoma, recognized as one of the most immunogenic malignancies in humans, holds paramount significance in the realm of immunotherapy. However, the emergence of drug resistance and the occurrence of adverse drug reactions underscore the pressing need to explore increasingly personalized immunotherapeutic modalities. Extracellular Vesicles (EVs), pivotal derivatives of immune cells, assume pivotal roles by encapsulating proteins, lipids, and nucleic acids within bilayer lipid structures, thereby facilitating targeted delivery to other immune cells. This orchestrated process orchestrates critical functions including antigen presentation, immune modulation, and the induction of apoptosis in tumor cells. A burgeoning body of evidence underscores the vast therapeutic potential of EVs in melanoma treatment. This comprehensive review aims to delineate the roles of EVs derived from immune cells such as dendritic cells, natural killer cells, macrophages, and T cells in the context of melanoma patients, thereby furnishing invaluable insights for the future direction of melanoma immunotherapy.

Effects of propofol-based total intravenous anesthesia versus desflurane anesthesia on natural killer cell cytotoxicity after hepatocellular carcinoma resection.

Background and Aims: Inhalation anesthesia suppresses the immune system and stimulates the growth of tumor cells, contrary to intravenous anesthesia. However, no consensus exists on which anesthetic technique is better for preventing cancer recurrence. Therefore, this study compared the effects of two different anesthetic techniques on natural killer cell cytotoxicity (NKCC) in hepatocellular carcinoma (HCC) patients undergoing open hepatic resection. Material and Methods: Patients diagnosed with nonmetastatic HCC were scheduled for hepatic resection and randomly assigned to receive either propofol- or desflurane-based anesthesia. The primary outcome was pre- and postoperative NKCC assay. Cytokine levels were assessed by measuring interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) levels, and the secondary outcome was postoperative cancer recurrence evaluated using diagnostic imaging scans for 2 years. Results: Twenty-eight patients were analyzed, including 15 and 13 in the total intravenous anesthesia (TIVA) and inhalation (INH) groups, respectively. Two patients in the INH group were excluded due to non-HCC postoperative pathologic results. At 24 h, the postoperative change in NKCC between both groups showed no significant differences at a ratio of effector cell: target cell = 1:1, 5:1, and 10:1 (P = 0.345, 0.345, and 0.565, respectively). Also, there were no significant differences in IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ levels (P = 0.588, 0.182, 0.730, 0.076, 0.518, 0.533, respectively). Postoperative tumor recurrence occurred in five and six patients in the TIVA and INH groups, respectively. Conclusion: NKCC did not differ significantly among HCC patients undergoing open hepatic resection under either propofol or desflurane anesthesia 24 h postoperatively.

A Self-Activating IL-15 Chimeric Cytokine Receptor to Empower Cancer Immunotherapy.

Background: Enhancing NK cells' antitumor activity requires sustained cytokine signaling. Interleukin-15 (IL-15) is a potent immunostimulatory cytokine used to armor CAR-NK and CAR-T cell immunotherapies. However, strategies to increase IL-15 expression and antitumor effect may trigger systemic toxicity with the potential to promote oncogenesis and autoimmune diseases. Methods: To overcome these limitations, we developed a new platform (IL15RB) whereby IL-15 with IL-2 signal peptide is tethered to its receptor, IL2Rß. Results: NK92-expressing IL15RB (NK92IL15RB) cells expand indefinitely without exogenous cytokines and have significantly higher anticancer activity than NK-92 stimulated by IL-15, IL-2, or expressing tethered IL-2. NK92IL5RB showed resistance to irradiation and IL-4. However, TGFß1 substantially reduced NK92IL5RB killing, suggesting the need to inhibit TGFß1 in IL-15-mediated immunotherapies. IL15RB induced strong STAT3 but weaker STAT5 and STAT1 activation compared to IL-2. Chronic exposure of NK92IL15RB cells to cancer cells reduced STAT3 and STAT1 activation irreversibly, suggesting a role in exhaustion. Combination with CAR-CD19 enhanced NK92IL15RB antitumor activity against leukemia and increased its STAT5 activation. NK92IL15RB anti-tumors activity was further enhanced by combination with anti-PD1. Conclusion: Our data suggest that the tethering of IL-15 to its receptor IL2Rß empowers NK cell cytolytic activity. Additionally, the tethering of IL-15 will prevent any systemic risk of toxicity.

Resting natural killer cells promote the progress of colon cancer liver metastasis by elevating tumor-derived stem cell factor.

The abundance and biological contribution of natural killer (NK) cells in cancer are controversial. Here, we aim to uncover clinical relevance and cellular roles of NK cells in colon cancer liver metastasis (CCLM). Here, we integrated single-cell RNA-sequencing, spatial transcriptomics (ST), and bulk RNA-sequencing datasets to investigate NK cells' biological properties and functions in the microenvironment of primary and liver metastatic tumors. Results were validated through an in vitro co-culture experiment based on bioinformatics analysis. Useing single-cell RNA-sequencing and ST, we mapped the immune cellular landscape of colon cancer and well-matched liver metastatic cancer. We discovered that GZMK+ resting NK cells increased significantly in tumor tissues and were enriched in the tumor regions of both diseases. After combining bulk RNA and clinical data, we observed that these NK cell subsets contributed to a worse prognosis. Meanwhile, KIR2DL4+ activated NK cells exhibited the opposite position and relevance. Pseudotime cell trajectory analysis revealed the evolution of activated to resting NK cells. In vitro experiments further confirmed that tumor-cell-co-cultured NK cells exhibited a decidual-like status, as evidenced by remarkable increasing CD9 expression. Functional experiments finally revealed that NK cells exhibited tumor-activating characteristics by promoting the dissociation of SCF (stem cell factor) on the tumor cells membrane depending on cell-to-cell interaction, as the supernatant of the co-culture system enhanced tumor progression. In summary, our findings revealed resting NK cells exhibited a clinical relevance with CCLM, which may be exploited for novel strategies to improve therapeutic outcomes for patients with CCLM.

Association of the Genetic Diversity of Killer Cell Immunoglobulin-like Receptor Genes and HLA-C Ligand in Saudi Women With Thyroid Cancer.

INTRODUCTION: Genetic diversity in the killer immunoglobulin-like receptor (KIR) gene composition and human leukocyte antigen (HLA) class I ligands, such as HLA-C, can affect the activity of natural killer cells and determine anti-cancer immunity. Specific KIR-HLA combinations can enhance cancer predisposition by promoting immune evasion. Studying the relationship between KIR-HLA polymorphisms and thyroid cancer (TC) risk can offer insights into how natural immunity fails, leading to disease development. Therefore, we investigated the association between KIR and HLA-C genotypes and TC risk in Saudi women. METHODS: In this retrospective study, sixteen KIR genotypes and 2 HLA-C allotypes were determined using the polymerase chain reaction-sequence-specific primer (PCR-SSP) method, and the genotypes of 50 Saudi female patients with TC were compared with those of 50 Saudi female healthy controls (HC). RESULTS: We observed a highly significant decrease in the presence of the KIR2DS2 and KIR2DS4 genes (OR = 0.15, 95% CI = 0.05-0.41, P = 0.0001; OR = 0.06, 95% CI = 0.02-0.2, P = 0.000, respectively) and in the presence of the KIR2DL5A gene (OR = 0.05, 95% CI = 0.02-0.14, P = 0.0000) in the TC group compared to the HC group. The frequency of the HLA-C2C2 allotype was significantly higher in HC compared to patients with TC (P = 0.02). The KIR haplotype group A and AB genotypes revealed a protective effect against TC (P = 0.0003 and P = 0.000, respectively), while the BB genotype showed a risk effect on TC compared to HC. Our results showed significant differences in the KIR gene combinations and KIR-HLA combinations between Saudi female TC patients and HC. CONCLUSION: These results suggest that the expression of KIR genes and their HLA-C ligands may influence the risk of TC development in Saudi women.

Genetic diversity in killer immunoglobulin-like receptors (KIR) gene composition and human leukocyte antigen class I (HLA) ligands such as HLA-C can impact the activity of natural killer cells (NK cells) and determine the results of cancer immunity. Specific KIR-HLA combinations can enhance vulnerability by promoting immune evasion. Studying the relationship between KIR-HLA polymorphisms and thyroid cancer (TC) risk can offer insights into how natural immunity failing leading to disease development. Therefore, we investigated the association between KIR and HLA-C genotypes and TC risk in Saudi women.

Effect of plasma-induced oxidation on NK cell immune checkpoint ligands: A computational-experimental approach.

Non-thermal plasma (NTP) shows promise as a potent anti-cancer therapy with both cytotoxic and immunomodulatory effects. In this study, we investigate the chemical and biological effects of NTP-induced oxidation on several key, determinant immune checkpoints of natural killer (NK) cell function. We used molecular dynamics (MD) and umbrella sampling simulations to investigate the effect of NTP-induced oxidative changes on the MHC-I complexes HLA-Cw4 and HLA-E. Our simulations indicate that these chemical alterations do not significantly affect the binding affinity of these markers to their corresponding NK cell receptor, which is supported with experimental read-outs of ligand expression on human head and neck squamous cell carcinoma cells after NTP application. Broadening our scope to other key ligands for NK cell reactivity, we demonstrate rapid reduction in CD155 and CD112, target ligands of the inhibitory TIGIT axis, and in immune checkpoint CD73 immediately after treatment. Besides these transient chemical alterations, the reactive species in NTP cause a cascade of downstream cellular reactions. This is underlined by the upregulation of the stress proteins MICA/B, potent ligands for NK cell activation, 24 h post treatment. Taken together, this work corroborates the immunomodulatory potential of NTP, and sheds light on the interaction mechanisms between NTP and cancer cells.

[Flow cytometry analysis of normal range of natural killer cells and their subsets in peripheral blood of healthy Chinese adults].

OBJECTIVE: To study the distribution characteristics of natural killer (NK) cells and their subsets in normal peripheral blood in China, and to explore their normal value and significance. METHODS: In this study, peripheral blood was collected from 200 healthy adults. Their age range was 18-87 years. All the subjects were divided into 6 age groups: 18-30, 31-40, 41-50, 51-60, 61-70, and 71-87 years. With CD16, CD56, CD4, CD19, as surface markers, fluid cytology detection techniques were used to detect NK cells and the relative and absolute counts. SPSS 27.0 was used for systematic analysis of the data, and the measurement data were expressed as mean±standard deviations. A t test, variance analysis or rank sum test were performed to compare the differences between the age groups and the sex groups. The significance level was set at α=0.05, and P < 0.05 was considered statistically significant. RESULTS: The range of NK B cells in the 200 healthy adult subjects was (0.46±0.24)×106/L, that of CD3-CD56+NK cells was (13.14±7.56)×106/L, that of CD56dimCD16+NK cells was (5.23±3.12)×106/L, that of CD56brightNK cells was (85.61±7.40)×106/L, and that of NK T cells was (4.16±3.34)×106/L. There were no statistically significant differences in CD3-CD56+NK cells and NK T cells with respect to age (P= 0.417, P=0.217). However, there was a decreasing trend in the number of NK B cells and CD56dimCD16+NK cells with increasing age (r=0.234, P < 0.001; r=0.099, P < 0.001), particularly after the age of 50. Conversely, CD56brightNK cells showed an increasing trend with age (r=0.143, P < 0.001). CONCLUSION: The detection of NK cells and their subsets has significant reference value for the diagnosis, treatment, and prognosis of autoimmune diseases, infectious diseases, and tumors. This study provides a preliminary reference range for clinical detection of NK cell subsets, but further research with a larger sample size and multi-center trials are needed to confirm these findings.

Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection were measured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus has great potential to modulate the TIME in PDAC.

CAR immunotherapy in autoimmune diseases: promises and challenges.

In recent years, the use of chimeric antigen receptor (CAR)-T cells has emerged as a promising immunotherapy in multiple diseases. CAR-T cells are T cells genetically modified to express a surface receptor, known as CAR, for the targeting of cognate antigens on specific cells. The effectiveness of CAR-T cell therapy in hematologic malignancies including leukemia, myeloma, and non-Hodgkin's lymphoma has led to consider its use as a potential avenue of treatment for autoimmune diseases. However, broadening the use of CAR-T cell therapy to a large spectrum of autoimmune conditions is challenging particularly because of the possible development of side effects including cytokine release syndrome and neurotoxicity. The design of CAR therapy that include additional immune cells such as double-negative T cells, γδ T cells, T regulatory cells and natural killer cells has shown promising results in preclinical studies and clinical trials in oncology, suggesting a similar potential utility in the treatment of autoimmune diseases. This review examines the mechanisms, efficacy, and safety of CAR approaches with a focus on their use in autoimmune diseases including systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, multiple sclerosis, myasthenia gravis, lupus nephritis and other autoimmune diseases. Advantages and disadvantages as compared to CAR-T cell therapy will also be discussed.

Engineering of a Multi-Modular DNA Nanodevice for Spatioselective Imaging and Evaluation of NK Cell-Mediated Cancer Immunotherapy.

Granzyme A (GzmA) secreted by natural killer (NK) cells has garnered considerable interest as a biomarker to evaluate the efficacy of cancer immunotherapy. However, current methodologies to selectively monitor the spatial distribution of GzmA in cancer cells during NK cell-targeted therapy are extremely challenging, primarily due to the existence of diverse cell populations, the low levels of GzmA expression, and the limited availability of GzmA probes. Herein we develop a multi-modular, structurally-ordered DNA nanodevice for evaluating NK cell-mediated cancer immunotherapy (MODERN), that permits spatioselective imaging of GzmA in cancer cells through GzmA-induced apurinic/apyrimidinic endonuclease 1 (APE1) inactivation. The MODERN incorporates multiple functional modules, including an APE1-gated recognition module, a photo-activated amplification module, an aptamer-mediated tumor-target module, and a polycatenane DNA module, enabling improved sensitivity and specificity towards intracellular GzmA. The MODERN was activated (on) in cancer cells due to the overexpression of APE1, whereas it remained silent (off) in the NK-treated cancer cells owing to the GzmA-induced APE1 inactivation. Furthermore, we demonstrated that GzmA-induced APE1 inactivation blocks the cellular repair of target cells, resulting in efficient cell death. This MODERN that relies on the specific inactivation of APE1 by GzmA should be beneficial for evaluating the efficacy of cancer immunotherapy.

An improved approach to generate IL-15+/+/TGFßR2-/- iPSC-derived natural killer cells using TALEN.

We present a TALEN-based workflow to generate and maintain dual-edited (IL-15+/+/TGFßR2-/-) iPSCs that produce enhanced iPSC-derived natural killer (iNK) cells for cancer immunotherapy. It involves using a cell lineage promoter for knocking in (KI) gene(s) to minimize the potential effects of expression of any exogenous genes on iPSCs. As a proof-of-principle, we KI IL-15 under the endogenous B2M promoter and show that it results in high expression of the sIL-15 in iNK cells but minimal expression in iPSCs. Furthermore, given that it is known that knockout (KO) of TGFßR2 in immune cells can enhance resistance to the suppressive TGF-ß signaling in the tumor microenvironment, we develop a customized medium containing Nodal that can maintain the pluripotency of iPSCs with TGFßR2 KO, enabling banking of these iPSC clones. Ultimately, we show that the dual-edited IL-15+/+/TGFßR2-/- iPSCs can be efficiently differentiated into NK cells that show enhanced autonomous growth and are resistant to the suppressive TGF-ß signaling.

Lipid-Based Nanoparticles Fused with Natural Killer Cell Plasma Membrane Proteins for Triple-Negative Breast Cancer Therapy.

Immunotherapy combined with chemicals and genetic engineering tools is emerging as a promising strategy to treat triple-negative breast cancer (TNBC), which is more aggressive with poorer progress than other breast cancer subtypes. In this study, lipid-based nanoparticles (LNPs) possessed an NK cell-like function that could deliver tumor-specific therapeutics and inhibit tumor growth. LNPs fused with an NK cell membrane protein system (NK-LNP) have three main features: (i) hydrophilic plasmid DNA can inhibit TNBC metastasis when encapsulated within LNPs and delivered to cells; (ii) the lipid composition of LNPs, including C18 ceramide, exhibits anticancer effects; (iii) NK cell membrane proteins are immobilized on the LNP surface, enabling targeted delivery to TNBC cells. These particles facilitate the targeted delivery of HIC1 plasmid DNA and the modulation of immune cell functions. Delivered therapeutic genes can inhibit metastasis of TNBC and then induce apoptotic cell death while targeting macrophages to promote cytokine release. The anticancer effect is expected to be applied in treating various difficult-to-treat cancers with LNP fused with NK cell plasma membrane proteins, which can simultaneously deliver therapeutic chemicals and genes.

Mesenchymal Stem/Stromal cells in solid tumor Microenvironment: Orchestrating NK cell remodeling and therapeutic insights.

Mesenchymal stem/stromal cells (MSCs), originating from normal tissues, possess the capacity to home to tumor sites and differentiate into tumor-associated MSCs (TA-MSCs), which are instrumental in shaping an immunosuppressive milieu within tumors. Natural killer (NK) cells, integral to the innate immune system, are endowed with the ability to eradicate target cells autonomously, serving as an immediate defense against neoplastic growths. Nonetheless, within the tumor microenvironment (TME), NK cells often exhibit a decline in both their numerical presence and functionality. TA-MSCs have been shown to exert profound inhibitory effects on the functions of tumor-infiltrating immune cells, notably NK cells. Understanding the mechanisms by which TA-MSCs contribute to NK cell dysfunction is critical for the advancement of immune surveillance and the enhancement of tumoricidal responses. This review summarizes existing literature on NK cell modulation by TA-MSCs within the TME and proposes innovative strategies to augment antitumor immunity.

Immunogenicity risk assessment of empty capsids present in adeno-associated viral vectors using predictive innate immune responses.

Immunogenicity of gene therapy and the impacts on safety and efficacy are of increasing interest in the pharmaceutical industry. Unique structural aspects of gene therapy delivery vectors, such as adeno-associated viral (AAV) vectors, are expected to activate the innate immune system. The risk of innate immune activation is critical to understand due to the potential impacts on safety and on subsequent adaptive immune responses. In this study, we investigated the responses of key innate immune players-dendritic cells, natural killer (NK) cells, and the complement system-to AAV8 capsids. Immunogenicity risk was also predicted in the presence empty AAV capsids for AAV gene therapy. Compared to genome-containing "full" AAV8 capsids, empty AAV8 capsids more strongly induced proinflammatory cytokine production and migration by human and mouse dendritic cells, but the "full" capsid increased expression of co-stimulatory markers. Furthermore, in an NK cell degranulation assay, we found mixtures of empty and full AAV8 capsids to activate expression of TNF-α, IFN-γ, and CD107a more strongly in multiple NK cell populations compared to either capsid type alone. Serum complement C3a was also induced more strongly in the presence of mixed empty and full AAV8 capsid formulations. Risk for innate immune activation suggests the importance to determine acceptable limits of empty capsids. Immunogenicity risk assessment of novel biological modalities will benefit from the aforementioned in vitro innate immune activation assays providing valuable mechanistic information.

Nebulized Immunotherapy of Orthotopic Lung Cancer by Mild Magnetothermal-based Innate Immunity Activations.

Advances in adaptive immunity have greatly contributed to the development of cancer immunotherapy. However, its over-low efficacy and insufficient invasion of immune cells in the tumor tissue, and safety problems caused by cytokine storm, have seriously impeded further clinical application for solid tumor immunotherapy. Notably, the immune microenvironment of the lungs is naturally enriched with alveolar macrophages (AMs). Herein, we introduce a novel nebulized magnetothermal immunotherapy strategy to treat orthotopic lung cancer by using magnetothermal nanomaterial (Zn-CoFe2O4@Zn-MnFe2O4-PEG, named ZCMP), which can release iron ions via an acid/thermal-catalytic reaction to maximize the use of lung's immune environment through the cascade activations of AMs and natural killer (NK) cells. Nebulized administration greatly enhance drug bioavailability by localized drug accumulation at the lesion site. Upon mild magnetic hyperthermia, the released iron ions catalyze endogenous H2O2 decomposition to produce reactive oxygen species (ROS), which triggers the M1 polarization of AMs, and the resultant inflammatory cytokine IFN-ß, IL-1ß and IL-15 releases to activate c-Jun, STAT5 and GZMB related signaling pathways, promoting NK cells proliferation and activation. This innovative strategy optimally utilizes the lung's immune environment and shows excellent immunotherapeutic outcomes against orthotopic lung cancer.

Chronic lymphoproliferative disorder of natural killer cells-related neurolymphomatosis with severe autonomic dysfunction: a case report.

BACKGROUND: Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a rare disease characterized by a persistent increase in NK cells in peripheral blood and is generally asymptomatic. If present, symptoms may include fatigue, B symptoms (fever, night sweats, and unintentional weight loss), autoimmune-associated diseases, splenomegaly, and infection due to neutropenia. Peripheral neuropathy, however, is uncommon with an incidence of 3%. Neurolymphomatosis is a neurological manifestation of non-Hodgkin lymphoma and leukemia in which neurotropic neoplastic cells infiltrate the nerves. Moreover, neurolymphomatosis caused by CLPD-NK is extremely rare, with even fewer cases of autonomic dysfunction. We report a case of neurolymphomatosis associated with CLPD-NK and developed autonomic dysfunction, including orthostatic hypotension and gastrointestinal symptoms. CASE PRESENTATION: The patient was a 61-year-old male who was referred to our hospital for leukocytosis. He was diagnosed with CLPD-NK; however, was untreated since he had no hepatosplenomegaly, and other systemic symptoms. He later developed numbness in his lower extremities. Cerebral spinal fluid examination revealed a markedly elevated protein level of 140 mg/dL, and contrast-enhanced magnetic resonance imaging showed bilateral L4 and 5 nerve roots with enlargement and contrast effect. An immune-mediated polyradiculoneuropathy was suspected, and he was treated with intravenous methylprednisolone and immunoglobulin followed by oral prednisolone and cyclosporine. Although his symptoms were relieved by the immunotherapy, significant autonomic dysfunction, including intractable diarrhea, decreased sweating, and orthostatic hypotension, appeared. Additionally, tests for onconeuronal antibodies, ganglionic nicotinic acetylcholine receptor (gAChR) antibody, NF155, CNTN1, Caspr1 antibody, and anti-ganglioside antibodies were all negative. A sural nerve biopsy revealed lymphocytic infiltration, and immunohistochemical staining of lymphocytes confirmed the infiltration of NK and T cells. Therefore, a diagnosis of neurolymphomatosis caused by CLPD-NK was made, and chemotherapy led to partial symptom improvement. CONCLUSIONS: We experienced a case of pathologically diagnosed neurolymphomatosis with autonomic dysfunction associated with CLPD-NK. In cases of subacute to chronic autonomic dysfunction, paraneoplastic neuropathy, amyloidosis, and autoimmune autonomic ganglionopathy are considered; however neurolymphomatosis caused by CLPD-NK, an important cause of autonomic dysfunction, is not. In difficult to make diagnosis, aggressive nerve biopsy is required.

Natural killer (NK) cells-related gene signature reveals the immune environment heterogeneity in hepatocellular carcinoma based on single cell analysis.

The early diagnosis of liver cancer is crucial for the treatment and depends on the coordinated use of several test procedures. Early diagnosis is crucial for precision therapy in the treatment of the hepatocellular carcinoma (HCC). Therefore, in this study, the NK cell-related gene prediction model was used to provide the basis for precision therapy at the gene level and a novel basis for the treatment of patients with liver cancer. Natural killer (NK) cells have innate abilities to recognize and destroy tumor cells and thus play a crucial function as the "innate counterpart" of cytotoxic T cells. The natural killer (NK) cells is well recognized as a prospective approach for tumor immunotherapy in treating patients with HCC. In this research, we used publicly available databases to collect bioinformatics data of scRNA-seq and RNA-seq from HCC patients. To determine the NK cell-related genes (NKRGs)-based risk profile for HCC, we isolated T and natural killer (NK) cells and subjected them to analysis. Uniform Manifold Approximation and Projection plots were created to show the degree of expression of each marker gene and the distribution of distinct clusters. The connection between the immunotherapy response and the NKRGs-based signature was further analyzed, and the NKRGs-based signature was established. Eventually, a nomogram was developed using the model and clinical features to precisely predict the likelihood of survival. The prognosis of HCC can be accurately predicted using the NKRGs-based prognostic signature, and thorough characterization of the NKRGs signature of HCC may help to interpret the response of HCC to immunotherapy and propose a novel tumor treatment perspective.