Cancer, being one of the most outrageous diseases, contributed to 48 % of the mortality in 2022, with lung cancer leading the race with a 12.4 % incidence rate. Conventional treatment modalities like radio-, chemo-, photo-, and immunotherapy employing nanocarriers often face several setbacks, such as non-specific delivery, off-site toxicity, rapid opsonization via the host immune system, and greater tumor recurrence rates. Moreover, the heterogeneous variability in the tumor microenvironment is responsible for existing therapy failure. With the advent of biomimetic nanoparticles as a novel and intriguing platform, researchers have exploited the inherent functionalities of the Cluster of Differentiation proteins (CD) as cell surface biomarkers and imparted the nanocarriers with enhanced homologous tumor targetability, immune evasion capability, and stealth properties, paving the way for improved therapy and diagnosis. This article explores pathogenesis and the multifaceted role of immune cells in non-small cell lung cancer. Moreover, the agenda of this article is to shed light on biomimetic nanoarchitectonics with respect to their fabrication, evaluation, and applications unraveling their synergistic effect with conventional therapies. Further discussion mentions the hurdles in clinical translation with viable solutions. The regulatory bottlenecks underscore the need for a regulatory roadmap with respect to commercialization. We believe that biomimetic nanoarchitectonics will be a beacon of hope in warfare against lung cancer.

The impact of cytomegalovirus (CMV) infection in shaping natural killer (NK) cell receptor (NKR) repertoire highlights the importance of NKRs in immunity against CMV. NKR-P1A (CD161) is an inhibitory NKR, whose expression is lost during CMV infection, but its role in NK cell responses during CMV infection is not known. Here, we show selective expansion of adaptive NKG2C+ NK cells lacking NKR-P1A receptor (NKR-P1A‒) due to their increased activation and proliferation compared with NKR-P1A+ NK cells in CMV-infected individuals. In vitro stimulation of PBMCs showed similar inherent proliferative capacity in both NKR-P1A+ versus NKR-P1A‒ NK cells in steady state and upregulation, but not loss of NKR-P1A receptor expression, in sorted NK cells. Furthermore, CMV infection induced differential gene expression profiles in NKR-P1A+ versus NKR-P1A‒ NK cells, and only NKR-P1A‒ NK cells exhibited transcriptome signatures associated with adaptive NK cells in CMV-infected individuals. This study further highlights the impact of CMV infection in shaping NK cell receptor repertoire and exclusion of NK cells that express the NKR-P1A receptor from the adaptive NKG2C+ NK cell population that expands during CMV infection.

Natural killer (NK) play a key role in controlling tumor dissemination by mediating cytotoxicity towards cancer cells without the need of education. These cells are pivotal in eliminating circulating tumor cells (CTCs) from the bloodstream, thus limiting cancer spread and metastasis. However, aggressive CTCs can evade NK cell surveillance, facilitating tumor growth at distant sites. In this review, we first discuss the biology of NK cells, focusing on their functions within the tumor microenvironment (TME), the lymphatic system, and circulation. We then examine the immune evasion mechanisms employed by cancer cells to inhibit NK cell activity, including the upregulation of inhibitory receptors. Finally, we explore the clinical implications of monitoring circulating biomarkers, such as NK cells and CTCs, for therapeutic decision-making and emphasize the need to enhance NK cell-based therapies by overcoming immune escape mechanisms.

Ulcerative colitis (UC) is a chronic, non-specific inflammatory bowel disease characterized by inflammation and injury of the colonic mucosa, exhibiting an increasing global incidence. Although research into UC pathogenesis is ongoing, the precise mechanisms remain to be fully elucidated. Studies indicate that UC development results from a complex interplay of factors, including genetic predisposition, environmental exposures, gut microbial dysbiosis, and immune dysregulation. Specifically, UC pathogenesis involves aberrant immune responses triggered by interactions between the host and gut microbiota. A complex, dynamic relationship exists between the microbial community and the host immune system throughout UC pathogenesis. Accumulating evidence suggests that changes in microbiota composition significantly impact gut immunity. This review will examine the intricate balance between the gut microbiota and mucosal immunity in UC progression and discuss potential therapeutic applications, providing a reference for further clinical treatment of this patient population.

Cellular senescence, a state of stable cell cycle arrest, acts as a double-edged sword in cancer biology. In young organisms, it acts as a barrier against tumorigenesis, but in the aging population, it may facilitate tumor growth and metastasis through the senescence-associated secretory phenotype (SASP). Natural killer (NK) cells play a critical role in the immune system, particularly in the surveillance, targeting, and elimination of malignant and senescent cells. However, age-related immunosenescence is characterized by declining NK cell function resulting in diminished ability to fight infection, eliminate senescent cells and suppress tumor development. This implies that preserving or augmenting NK cell function may be central to defense against age-related degenerative and malignant diseases. This review explores the underlying mechanisms behind these interactions, focusing on how aging influences the battle between the immune system and cancer, the implications of senescent NK cells in disease progression, and the potential of adoptive NK cell therapy as a countermeasure to these age-related immunological challenges.

Wound healing, a physiological process, involves several different types of cells, from immune cells to non-immune cells, including mesenchymal stromal/stem cells (MSC), and their interactions. Immune cells including macrophages, neutrophils, dendritic cells (DC), innate lymphoid cells (ILC), natural killer (NK) cells, and B and T lymphocytes participate in wound healing by secreting various mediators and interacting with other cells. MSCs, as self-renewing, fast proliferating, and multipotent stromal/stem cells, are found in a wide variety of tissues and critically involved in different phases of wound healing by secreting various molecules that help to improve tissue healing and regeneration. In this review, first, we described the four main phases of wound healing, second, we reviewed the function of MSCs, MSC secretome and immune cells in improving the progress of wound repair (mainly focusing on skin wound healing), third, we explained the immune cells/MSCs interactions in the process of wound healing and regeneration, and finally, we introduce clinical applications of MSCs to improve the process of wound healing.

Cerebral arteriovenous malformation is a congenital blood vessel abnormality with its immune mechanism remains unclear. Our study characterized the change of cellular composition and gene expression landscape in brain arteriovenous malformation (bAVM).

We conducted single-cell RNA sequencing analysis on one bAVM sample and three healthy control (HC) samples. Cell clustering analysis and cell type annotation were used to identify the major cell types in bAVM and HC samples. Critical differentially expressed genes between bAVM and HC sample were analyzed in each cell types to explore the functional changes of each kind of cells. We also examined the cell communication change in bAVM sample and identified the significantly changed cellular interaction pathways.

5 major cell types were identified including NK cells, monocytes, fibroblasts, endothelial cells (EC), tissue stem cells and smooth muscle cells (SMC). In bAVM sample, proportion of monocytes raised significantly while SMC decreased. Inflammation and cell migration related genes expression and cell communication pathways changed dramatically in bAVM sample.

Inhibition of monocyte-endothelium interaction and promotion of NK cells interaction were found in bAVM sample, which may reveal a new mechanism about inflammation response and cellular impairment in the disease progression.

Chimeric antigen receptor (CAR) NK cell therapy has emerged as a promising alternative to CAR T cell therapy, offering significant advantages in terms of safety and versatility. Here we explore the current clinical landscape of CAR NK cells, and their application in hematologic malignancies and solid cancers, as well as their potential for treating autoimmune disorders. Our analysis draws from data collected from 120 clinical trials focused on CAR NK cells, and presents insights into the demographics and characteristics of these studies. We further outline the specific targets and diseases under investigation, along with the major cell sources, genetic modifications, combination strategies, preconditioning- and dosing regimens, and manufacturing strategies being utilized. Initial results from 16 of these clinical trials demonstrate promising efficacy of CAR NK cells, particularly in B cell malignancies, where response rates are comparable to those seen with CAR T cells but with lower rates of severe adverse effects, such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and graft-versus-host disease (GvHD). However, challenges remain in solid tumor applications, where only modest efficacy has been observed to date. Our analysis reveals that research is increasingly focused on enhancing CAR NK cell persistence, broadening their therapeutic targets, and refining manufacturing processes to improve accessibility and scalability. With recent advancements in NK cell engineering and their increased clinical applications, CAR NK cells are predicted to become an integral component of next-generation immunotherapies, not only for cancer but potentially for immune-mediated diseases as well.

Ovarian cancer is a lethal disease with low survival rates for women diagnosed in advanced stages. Current cancer immunotherapies are not efficient in ovarian cancer, and there is therefore a significant need for novel treatment options. The β-galactoside-binding lectin, Galectin-3, is involved in different immune processes and has been associated with poor outcome in various cancer diagnoses. Here, we investigated how Galectin-3 affects the interaction between natural killer (NK) cells and neutrophils in the tumor microenvironment of ovarian cancer.

Ascites from the metastatic tumor microenvironment and cyst fluid from the primary tumor site were collected from patients with high-grade serous carcinoma (HGSC) together with peripheral blood samples. Galectin-3 concentration was measured in ascites, cyst fluid and serum or plasma. Neutrophils isolated from HGSC ascites and autologous blood were analyzed to evaluate priming status and production of reactive oxygen species. In vitro co-culture assays with NK cells, neutrophils and K562 target cells (cancer cell line) were conducted to evaluate NK cell viability, degranulation and cytotoxicity.

High levels of Galectin-3 were observed in cyst fluid and ascites from patients with HGSC. Neutrophils present in HGSC ascites showed signs of priming; however, the priming status varied greatly among the patient samples. Galectin-3 induced production of reactive oxygen species in ascites neutrophils, but only from a fraction of the patient samples, which is in line with the heterogenous priming status of the ascites neutrophils. In co-cultures with NK cells and K562 target cells, we observed that Galectin-3-induced production of reactive oxygen species in neutrophils resulted in decreased NK cell viability and lowered anti-tumor responses.

Taken together, our results demonstrate high levels of Galectin-3 in the tumormicroenvironment of HGSC. High levels of Galectin-3 may induce production of reactiveoxygen species in ascites neutrophils in some patients. In turn, reactive oxygen species produced by neutrophils may modulate the NK cell anti-tumor immunity. Together, this study suggests further investigation to evaluate if a Galectin-3-targeting therapy may be used in ovarian cancer.

In 2024, the United States was projected to experience 2 million new cancer diagnoses and approximately 611,720 cancer-related deaths, reflecting a broader global trend in which cancer cases are anticipated to exceed 35 million by 2050. This increasing burden highlights ongoing challenges in cancer treatment despite significant advances that have reduced cancer mortality by 31% since 1991. Key obstacles include the disease's inherent heterogeneity and complexity, such as treatment resistance, cancer stem cells, and the multifaceted tumor microenvironment (TME). The TME-comprising various tumor and immune cells, blood vessels, and biochemical factors-plays a crucial role in tumor growth and resistance to therapies. Recent innovations in cancer treatment, particularly in the field of immuno-oncology, have leveraged insights into TME interactions. An emerging example is the FDA-approved therapy using tumor-infiltrating lymphocytes (TILs), demonstrating the potential of cell-based approaches in solid tumors. However, TIL therapy is just one of many strategies being explored. This review provides a comprehensive overview of the emerging field of immuno-oncology, focusing on how novel therapies targeting or harnessing components of the TME could enhance treatment efficacy and address persistent challenges in cancer care.

Natural killer (NK) cells have multifaceted roles within the complex tumor milieu. They are pivotal components of innate immunity and shape the dynamic landscape of tumor-immune cell interactions, and thus can be leveraged for use in therapeutic interventions. NK-based immunotherapies have had remarkable success in hematological malignancies, but these therapies are met with many challenges in solid tumors, including neuroblastoma (NB), a childhood tumor arising from the sympathetic nervous system. With a focus on NB, this review outlines the mechanisms employed by NK cells to recognize and eliminate malignant cells, delving into the dynamic relationship between ligand-receptor interactions, cytokines, and other molecules that facilitate the cross talk between NK and NB cells. We discuss the immunomodulatory functions of NK cells and the mechanisms that contribute to loss of this immunosurveillance in NB, with a focus on how this dynamic has been utilized in recent immunotherapy advancements for NB.

Human memory NK cells represent a heterogeneous CD56dim population that expands and persists in human cytomegalovirus (HCMV)-seropositive healthy individuals. They are characterized by the preferential, not fully overlapping, expression of NKG2C (activating receptor for HLA-E) and CD57 maturation marker, and by the lack of FcεRIγ adaptor chain. Hyperresponsiveness to Fcγ receptor IIIA (CD16) engagement represents the distinctive functional signature of memory NK cells. Although CD16 engagement was shown to acutely enhance glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet.

Here, we describe the peculiar nutrient transporter expression pattern of FcεRIγ- memory NK cells, characterized by higher levels of CD98 neutral amino acid antiporter and CD71 transferrin receptor, and lower expression of GLUT1 glucose transporter, with respect to FcεRIγ+ conventional NK cells. Although CD16 engagement acutely enhances glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet. Our results firstly show that sustained CD16 engagement by contact with IgG-opsonized target cells induces the mTORC1-dependent upregulation of CD98 and CD71 nutrient receptors on CD56dim NK cells, in a transporter-specific fashion, that is finely tuned by cell-dependent (grade of functional maturation, and memory or conventional lineage) and stimulus-dependent (time length and cooperation with cytokines) factors. We also demonstrate that CD98 antiporter function is required for CD16-dependent IFN-γ production, and that enhanced CD98-mediated neutral amino acid uptake associates with heightened memory NK cell functional response.

Collectively, our work documents that CD16 engagement leads to a metabolic rewiring of human NK cells and suggests that a distinct nutrient transporter expression pattern may contribute to memory NK cell peculiar functional features.

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer related deaths. Targeting neuroblastoma with immunotherapies has proven challenging due to a paucity of immune cells in the tumor microenvironment and the release of immunosuppressive cytokines by neuroblastoma tumor cells. We hypothesized that combining an oncolytic Herpes Simplex Virus (oHSV) with natural killer (NK) cells might overcome these barriers and incite tumor cell death.

We utilized MYCN amplified and non-amplified neuroblastoma cell lines, the IL-12 expressing oHSV, M002, and the human NK cell line, NK-92 MI. We assessed the cytotoxicity of NK cells against neuroblastoma with and without M002 infection, the effects of M002 on NK cell priming, and the impact of M002 and priming on the migratory capacity and CD107a expression of NK cells. To test clinical applicability, we then investigated the effects of M002 and NK cells on neuroblastoma in vivo.

NK cells were more attracted to neuroblastoma cells that were infected with M002. There was an increase in neuroblastoma cell death with the combination treatment of M002 and NK cells both in vitro and in vivo. Priming the NK cells enhanced their cytotoxicity, migratory capacity and CD107a expression.

To the best of our knowledge, these investigations are the first to demonstrate the effects of an oncolytic virus combined with self-maintaining NK cells in neuroblastoma and the priming effect of neuroblastoma on NK cells. The current studies provide a deeper understanding of the relation between NK cells and neuroblastoma and these data suggest that oHSV increases NK cell cytotoxicity towards neuroblastoma.

The objective of our study was to evaluate the effect of endophyte-infected tall fescue (E+) seeds intake on liver tissue transcriptome in growing Angus × Simmental steers and heifers through RNA-seq analysis. Normal weaned calves (~8 months old) received either endophyte-free tall fescue (E-; n = 3) or infected tall fescue (E+; n = 6) seeds for a 30-d period. The diet offered was ad libitum bermudagrass (Cynodon dactylon) hay combined with a nutritional supplement of 1.61 kg (DM basis) of E+ or E- tall fescue seeds, and 1.61 kg (DM basis) of energy/protein supplement pellets for a 30-d period. Dietary E+ tall fescue seeds were included in a rate of 20 μg of ergovaline/kg BW/day. Liver tissue was individually obtained through biopsy at d 30. After preparation and processing of the liver samples for RNA sequencing, we detected that several metabolic pathways were activated (i.e., upregulated) by the consumption of E+ tall fescue. Among them, oxidative phosphorylation, ribosome biogenesis, protein processing in endoplasmic reticulum and apoptosis, suggesting an active mechanism to cope against impairment in normal liver function. Interestingly, hepatic protein synthesis might increase due to E+ consumption. In addition, there was upregulation of "thermogenesis" KEGG pathway, showing a possible increase in energy expenditure in liver tissue due to consumption of E+ diet. Therefore, results from our study expand the current knowledge related to liver metabolism of growing beef cattle under tall fescue toxicosis.

Natural killer (NK) cells kill target cells following triggering via germline-encoded receptors interacting with target cell-expressed ligands (direct killing), or via antibody-dependent cellular cytotoxicity (ADCC) mediated by FcγRIIIa. NK cytotoxicity is modulated by signaling through activating or inhibitory receptors. A major checkpoint is mediated by the NK inhibitory receptor NKG2A/CD94 and its target cell ligand, HLA-E, which is complexed with HLA signal sequence-derived peptides termed VL9 (HLA-E-VL9). We have previously reported the isolation of a murine HLA-E-VL9-specific IgM antibody 3H4 and the generation of a higher affinity IgG version (3H4v3). Here we have used phage display library selection to generate a high affinity version of 3H4v3, called 3H4v31, with an ∼700 fold increase in binding affinity. We show using an HLA-E-VL9+ K562 tumor model that, in vitro, the addition of 3H4v31 to target cells increased direct killing of targets by CD16-negative NK cell line NK-92 and also mediated ADCC by NK-92 cells transfected with CD16. Moreover, ADCC by primary NK cells was also enhanced in vitro by 3H4v31. 3H4v31 was also able to bind and enhance target cell lysis of endogenously expressed HLA-E-VL9 on human cervical cancer and human pancreatic cancer cell lines. In vivo, 3H4v31 slowed the growth rate of HLA-E-VL9+ K562 tumors implanted into NOD/SCID/IL2rγ null mice compared to isotype control when injected with NK-92 cells intratumorally. Together, these data demonstrate that mAb 3H4v31 can enhance NK cell killing of HLA-E-VL9-expressing tumor cells in vitro by both direct killing activity and by ADCC. Moreover, mAb 3H4v31 can enhance NK cell control of tumor growth in vivo. We thus identify HLA-E-VL9 monoclonal antibodies as a promising novel anti-tumor immunotherapy.

A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing by checkpoint inhibition and antibody dependent cellular cytotoxicity.

Flow cytometry of blood samples is a very valuable clinical and research tool to monitor the immune response in human patients. Furthermore, it has been successfully applied in cats, such as for infections with feline immune deficiency virus (FIV). However, if cells are not isolated and frozen, analysis of anticoagulated blood samples requires mostly prompt processing following blood collection, making later analysis of stored full blood samples obtained in clinical studies often impossible. The SMART Tube system (SMART TUBE Inc., California, United States; SMT) allows fixation and long-term preservation of whole blood samples at -80°C. However, this system has so far only been applied to human biological samples. In the present study, a new flow cytometry SMART Tube protocol adapted for feline whole blood samples was successfully established allowing quantification of T-helper cells, cytotoxic T-cells, B-cells, monocytes, and neutrophils up to 2 years post sampling. Results obtained from frozen stabilized and fresh blood samples were compared for validation purposes and correlated to differential blood counts from a conventional hematology analyzer. Clinical applicability of the new technique was verified by using samples from a treatment study for feline infectious peritonitis (FIP). Using the new SMT protocol on retained samples, it could be demonstrated that long-term storage of these SMT tubes is also possible. In summary, the newly adapted SMT protocol proved suitable for performing flow cytometry analysis on stored feline whole blood samples, thus opening up new avenues for veterinary research on a variety of aspects of clinical interest.

Although the development of immunotherapies has been revolutionary in the treatment of several cancers, many cancer types remain unresponsive to immune-based treatment and are largely managed by chemotherapy drugs. However, chemotherapeutics are not infallible and are frequently rendered ineffective as resistance develops from prolonged exposure. Recent investigations have indicated that some chemotherapy drugs have additional functions beyond their normative cytotoxic capacity and are in fact immune-modifying agents. Of the pharmaceuticals with identified immune-editing properties, gemcitabine is well-studied and of interest to clinicians and scientists alike. Gemcitabine is a chemotherapy drug approved for the treatment of multiple cancers, including breast, lung, pancreatic, and ovarian. Because of its broad applications, relatively low toxicity profile, and history as a favorable combinatory partner, there is promise in the recharacterization of gemcitabine in the context of the immune system. Such efforts may allow the identification of suitable immunotherapeutic combinations, wherein gemcitabine can be used as a priming agent to improve immunotherapy efficacy in traditionally insensitive cancers. This review looks to highlight documented immunomodulatory abilities of one of the most well-known chemotherapy agents, gemcitabine, relating to its influence on cells and proteins of the immune system.

Our previous studies have introduced osteoclasts (OCs) as major activators of NK cells. It was found that OCs exhibit the capabilities of inducing cell expansion as well as increasing the cytotoxic activity of NK cells by granule release and increasing the secretion of TNF-α and TRAIL, leading to increased lysis of tumors in short-term as well as long-term periods, respectively. OC- induced expanded NK cells were named supercharged NK cells (sNK) due to their significantly high functional activity as well as their significantly higher cell expansion rate. It is, however, unclear whether the OC-mediated effect in NK cells is specific or whether other cytotoxic immune cells can also be expanded and activated by OCs. We chose to focus on γδ T cells and pan T cells, which also include CD8+ T cells. In this paper, we report that OCs are capable of expanding and functionally activating both γδ T cells and pan T cells. Expanded γδ T and pan T cells were capable of secreting high levels of INF-γ, albeit with different dynamics to those of NK cells, and, moreover, they are unable to kill NK-specific targets. Since we used humanized-BLT (hu-BLT) mice as a model of human disease, we next determined whether NK and T cell activation through OCs is also evident in cells obtained from hu-BLT mice. Similar to humans, OCs were capable of increasing the cell expansion and secretion of IFN-γ in the culture of either NK or T cells from hu-BLT mice, providing yet further evidence that these mice are appropriate models to study human disease. Therefore, these studies indicated that CD3+ T or γδ T cells can proliferate and be supercharged by OCs similar to the NK cells; thus, they can be used individually or in combination in the cell therapy of cancers.

Immune cell dysfunction within the tumor microenvironment (TME) undermines the control of cancer progression. Established tumors contain phenotypically distinct, tumor-specific natural killer (NK) cells; however, the temporal dynamics, mechanistic underpinning and functional significance of the NK cell compartment remains incompletely understood. Here, we use photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of intratumoral NK cells. We reveal that NK cells rapidly lose effector functions and adopt a distinct phenotypic state with features associated with tissue residency. NK cell depletion from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti-tumor responses. IL-15 administration prevented loss of function and improved tumor control, generating intratumoral NK cells with both tissue-residency characteristics and enhanced effector function. Collectively, our data reveals the fate of NK cells after recruitment into tumors and provides insight into how their function may be revived.

Cancer still represents the second leading cause of death right after cardiovascular diseases. According to the World Health Organization (WHO), cancer provoked around 10 million deaths in 2020, with lung and colon tumors accounting for the deadliest forms of cancer. As tumor cells become resistant to traditional therapeutic approaches, immunotherapy has emerged as a novel strategy for tumor control. T lymphocytes are key players in immune responses against tumors. Immunosurveillance allows identification, targeting and later killing of cancerous cells. Nevertheless, tumors evolve through different strategies to evade the immune response and spread in a process called metastasis. The ineffectiveness of traditional strategies to control tumor growth and expansion has led to novel approaches considering modulation of T cell activation and effector functions. Program death receptor 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) showed promising results in the early 90s and nowadays are still being exploited together with other drugs for several cancer types. Other negative regulators of T cell activation are diacylglycerol kinases (DGKs) a family of enzymes that catalyze the conversion of diacylglycerol (DAG) into phosphatidic acid (PA). In T cells, DGKα and DGKζ limit the PLCγ/Ras/ERK axis thus attenuating DAG mediated signaling and T cell effector functions. Upregulation of either of both isoforms results in impaired Ras activation and anergy induction, whereas germline knockdown mice showed enhanced antitumor properties and more effective immune responses against pathogens. Here we review the mechanisms used by DGKs to ameliorate T cell activation and how inhibition could be used to reinvigorate T cell functions in cancer context. A better knowledge of the molecular mechanisms involved upon T cell activation will help to improve current therapies with DAG promoting agents.

Some reports have indicated a high expression level of ASB3 in various cancers, but its role in hepatocellular carcinoma (HCC) remains elusive.

ASB3 levels and clinical features were obtained from the TCGA database. Meanwhile, the expression levels of ASB3 in tumor and paraneoplastic tissues were further verified by qRT-PCR and Imunohistochemistry (IHC). ASB3-related downstream molecular analysis was carried out with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Pathways linked to ASB3 expression were identified by means of gene set enrichment analysis (GSEA). Single-sample GSEA (ssGSEA) aided in conducting a correlation analysis of ASB3 with immune infiltration. Functional experiments were performed in HepG2 cells by using the small interfering RNA.

ASB3 expression was remarkably higher in HCC tissues. Its remarkable precision in forecasting cancer suggests that ASB3 might serve as an unidentified diagnostic and prognostic indicator of HCC. Higher ASB3 expression led to worse overall survival (OS), particularly in various clinical subgroups of HCC. GO/KEGG analysis indicated that critical biological activities, such as the activation of complement systems and humoral immune response, could potentially underlie the progression of HCC. Furthermore, GSEA demonstrated enrichment of certain pathways, including the MAPK, IL17, and fibrinolysis pathways, in samples with elevated ASB3 levels. ASB3 exhibited a substantial association with T helper cells, dendritic cells (DCs), and central memory T (Tcm) cell infiltration level. Cell function experiments confirmed elevated ASB3 levels in HCC cell lines as opposed to hepatic epithelial cell lines. Moreover, the ability of HCC cells to proliferate and invade was remarkably reduced by ASB3 knockdown.

Summarize briefly, we found that ASB3 can be a promising biomarker in HCC.

Studying the immune system in vitro aims to understand how, when, and where the immune cells migrate/differentiate and respond to the various triggering events and the decision points along the immune response journey. It becomes evident that organ-on-a-chip (OOC) technology has a superior capability to recapitulate the cell-cell and tissue-tissue interaction in the body, with a great potential to provide tools for tracking the paracrine signaling with high spatial-temporal precision and implementing in situ real-time, non-destructive detection assays, therefore, enabling extraction of mechanistic information rather than phenotypic information. However, despite the rapid development in this technology, integration of the immune system into OOC devices stays among the least navigated tasks, with immune cells still the major missing components in the developed models. This is mainly due to the complexity of the immune system and the reductionist methodology of the OOC modules. Dedicated research in this field is demanded to establish the understanding of mechanism-based disease endotypes rather than phenotypes. Herein, we systemically present a synthesis of the state-of-the-art of immune-cantered OOC technology. We comprehensively outlined what is achieved and identified the technology gaps emphasizing the missing components required to establish immune-competent OOCs and bridge these gaps.

The anti-tumor effects of natural killer (NK) cells vary among individuals. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressed on liver NK cells is a marker of anti-tumor cytotoxicity against hepatocellular carcinoma (HCC) in immune cell therapy. This study aimed to develop a liver immune status index (LISI) that predicts low TRAIL expression and validates its ability to predict recurrence after initial hepatectomy for primary HCC.

A functional analysis of liver NK cells co-cultured with interleukin-2 for 3 days was performed of 40 liver transplant donors. The LISI, which predicted low TRAIL expression (25% quartile: <33%) in liver NK cells, was calculated using multiple logistic regression analysis. Next, 586 initial hepatectomy cases were analyzed based on the LISI.

Our model was based on the Fibrosis-4 index+0.1 (odds ratio [OR], 1.33), body mass index (OR, 0.61), and albumin levels+0.1 (OR, 0.54). The area under the receiver operating characteristic curve (AUC) of the LISI for low TRAIL expression was 0.89. Stratification of the recurrence rates (RR) revealed that LISI was an independent predictive factor of RR (moderate risk: hazard ratio, 1.44; high risk: hazard ratio, 3.02). The AUC was similar for the LISI, albumin-indocyanine green evaluation grade, albumin-bilirubin score, and geriatric nutritional risk index for predicting RR. Among the vascular invasion cases, the LISI was more useful than the other indexes.

Our model facilitates the prediction of RR in high-risk patients by providing LISI to predict the anti-tumor effects of NK cells.

Oridonin is a Chinese herbal medicine exhibiting anti-tumor properties; however, its immune modulation capacity has yet to be elucidated. Our objective in this study was to determine whether oridonin enhances the anti-tumor activity of natural killer (NK) cells against lung cancer cells.

LDH-releasing assays were used to investigate the effects of oridonin on NK-92MI cell activity against lung cancer cells. Flow cytometry and real-time PCR were used to examine the effects of oridonin on degranulation markers, cytotoxic factors, activating receptors on NK-92MI cells, and ligands in lung cancer cells. Western blot analysis provided insight into the mechanisms underlying the observed effects.

Oridonin enhanced the cytotoxic effects of NK-92MI cells against A549 lung cancer cells. This effect involved upregulating the expression of the degranulation marker CD107a and IFN-γ as well as activating receptors on NK cells and their ligand MICA/B. Oridonin also inhibited STAT3 phosphorylation in A549 cells and NK-92MI cells. A lung cancer mouse model confirmed the anti-tumor effects of oridonin and NK-92MI cells, wherein both treatments alone suppressed tumor growth. Oridonin was also shown to have a synergistic effect on the anti-tumor activity of NK-92MI cells.

The ability of oridonin to enhance the cytotoxic effects of NK cells indicates its potential as a novel therapeutic agent for the treatment of lung cancer.

To identify, quantify, and characterize leukocyte populations in PI and periodontitis using flow cytometry.

Fresh biopsies from human PI and periodontitis lesions were processed to a single-cell suspension. The immune cell types were identified using flow cytometry.

Twenty-one biopsies were obtained and analyzed corresponding to fourteen PI and seven periodontitis samples. Participants' average age was 63.95 ± 14.77 years without a significant difference between PI and periodontitis patients, the female/male ratio was 8/12, and mean PD was 8.5 ± 2.17. High similarity was found between periodontitis and PI in the main immune cell types. Out of the leukocytes, the PMN proportion was 40% in PI and 33% in periodontitis. T-cells 22% in PI and 18% in periodontitis. Similar proportions of B-cells 10% and macrophages 6% were found in PI and periodontitis. Dendritic and NK cells were found in low proportions (~ 1%) in PI and periodontitis. T-cell sub-analysis showed that CD4-positive were more prevalent than CD8-positive in both diseases (CD4/CD8 ratio of 1.2).

With the use of flow cytometry analysis, the leukocyte populations in human peri-implantitis and periodontitis were classified. In PI and periodontitis, we identified similar proportions of specific (CD4/CD8) and innate (dendritic and NK) immune cells. These results corroborate previous histological studies.

Flow cytometry analysis can be used to identify and quantify immune cells in PI and periodontitis, including sub-classification of T cells (CD4/8) as well as detection of cells that require multiple markers for identification (such as dendritic cells).

Ducks have recently received a lot of attention from the research community due to their importance as natural reservoirs of avian influenza virus (AIV). Still, there is a lack of tools to efficiently determine the immune status of ducks. The purpose of this work was to develop an automated differential blood count for the mallard duck (Anas platyrhynchos), to assess reference values of white blood cell (WBC) counts in this species, and to apply the protocol in an AIV field study. We established a flow cytometry-based duck WBC differential based on a no-lyse no-wash single-step one-tube technique, applying a combination of newly generated monoclonal antibodies with available duck-specific as well as cross-reacting chicken markers. The blood cell count enables quantification of mallard thrombocytes, granulocytes, monocytes, B cells, CD4+ T cells (T helper) and CD8+ cytotoxic T cells. The technique is reproducible, accurate, and much faster than traditional evaluations of blood smears. Stabilization of blood samples enables analysis up to 1 week after sampling, thus allowing for evaluation of blood samples collected in the field. We used the new technique to investigate a possible influence of sex, age, and AIV infection status on WBC counts in wild mallards. We show that age has an effect on the WBC counts in mallards, as does sex in juvenile mallards. Interestingly, males naturally infected with low pathogenic AIV showed a reduction of lymphocytes (lymphocytopenia) and thrombocytes (thrombocytopenia), which are both common in influenza A infection in humans. IMPORTANCE Outbreaks of avian influenza in poultry and humans are a global public health concern. Aquatic birds are the primary natural reservoir of avian influenza viruses (AIVs), and strikingly, AIVs mainly cause asymptomatic or mild infection in these species. Hence, immunological studies in aquatic birds are important for investigating variation in disease outcome of different hosts to AIV and may aid in early recognition and a better understanding of zoonotic events. Unfortunately, immunological studies in these species were so far hampered by the lack of diagnostic tools. Here, we present a technique that enables high-throughput white blood cell (WBC) analysis in the mallard and report changes in WBC counts in wild mallards naturally infected with AIV. Our protocol permits large-scale immune status monitoring in a widespread wild and domesticated duck species and provides a tool to further investigate the immune response in an important reservoir host of zoonotic viruses.

Cutaneous T-cell lymphoma (CTCL) is an incurable and cosmetically disfiguring disease associated with microenvironmental signals. We investigated the effects of CD47 and PD-L1 immune checkpoint blockades, as a strategy for targeting both innate and adaptive immunity. CIBERSORT analysis identified the immune-cell composition in the CTCL tumor microenvironment and the immune checkpoint expression profile for each immune-cell gene cluster from CTCL lesions. We investigated the relationship between MYC and CD47 and PD-L1 expression and found that MYC short hairpin RNA knockdown and MYC functional suppression by TTI-621 (SIRPαFc) and anti-PD-L1 (durvalumab) in CTCL cell lines reduced the expression of CD47 and PDL1 mRNA and protein as measured by qPCR and flow cytometry, respectively. In vitro, blockade of the CD47-SIRPα interaction with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8+ T-cell-mediated killing in a mixed leucocyte reaction. Moreover, TTI-621 synergized with anti-PD-L1 in macrophages reprogram to M1-like phenotypes and inhibited CTCL cell growth. These effects were mediated by cell death-related pathways, including apoptosis, autophagy, and necroptosis. Collectively, our findings show that CD47 and PD-L1 are critical regulators of immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy for CTCL.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide. Both genetic and environmental factors through a multitude of underlying molecular mechanisms participate in the pathogenesis of HCC. Recently, numerous studies have shown that circular RNAs (circRNAs), an emerging class of non-coding RNAs characterized by the presence of covalent bonds linking 3' and 5' ends, play an important role in the initiation and progression of cancers, including HCC. In this review, we outline the current status of the field of circRNAs, with an emphasis on the functions and mechanisms of circRNAs in HCC and its microenvironment. We also summarize and discuss recent advances of circRNAs as biomarkers and therapeutic targets. These efforts are anticipated to throw new insights into future perspectives about circRNAs in basic, translational and clinical research, eventually advancing the diagnosis, prevention and treatment of HCC.

Natural killer (NK) cells play a crucial role in early immune defenses against transformed cells and are used in the therapeutic management of cancer. However, it is difficult to sufficiently obtain high purity activated NK cells for clinical application. The function of NK cells is dependent on the balance of activating and inhibitory signals. Strong and diverse stimuli are required to increase the function of NK cells. Radiotherapy modulates the expression of various immunomodulatory molecules that recruit and activate NK cells. NK cell-mediated antibody-dependent cellular cytotoxicity is one of the most potent cytotoxic effects of NK cells against target cancer cells. To generate activated and irradiated autologous peripheral blood mononuclear cells (PBMCs), cytokine and monoclonal antibody stimulation followed by ionizing radiation was performed in the present study. The expanded NK cells were cultured for 21 days using activated/irradiated autologous PBMCs. Colorectal cancer cells (SW480 and HT-29) were used to analyze the expression of NK group 2D ligands and EGFR by radiation. The cytotoxicity of radiation plus NK cell-based targeted therapy against colorectal cancer cell lines was analyzed using flow cytometry. Activated and irradiated PBMCs exhibited significantly increased expression of various activating ligands that stimulated NK cells. In total, >10,000-fold high-purity activated NK cells were obtained, with negligible T-cell contamination. To confirm the antitumor activity of the NK cells expanded by this method, the expanded NK cells were treated with cetuximab, radiotherapy, or a combination of cetuximab and radiotherapy in the presence of human colorectal cancer cells. Expanded NK cells were effective at targeting human colorectal cancer cells, particularly when combined with cetuximab and radiotherapy. Thus, in the present study, a novel method for high-purity activated NK cell expansion was developed using activated and irradiated PBMCs. In addition, combined radiotherapy and antibody-based immunotherapy with expanded NK cells may be an effective strategy to enhance the efficiency of treatment against colorectal cancer.

Exponential accumulation of single-cell transcriptomes poses great challenge for efficient assimilation. Here, we present an approach entitled generative pretraining from transcriptomes (tGPT) for learning feature representation of transcriptomes. tGPT is conceptually simple in that it autoregressive models the ranking of a gene in the context of its preceding neighbors. We developed tGPT with 22.3 million single-cell transcriptomes and used four single-cell datasets to evalutate its performance on single-cell analysis tasks. In addition, we examine its applications on bulk tissues. The single-cell clusters and cell lineage trajectories derived from tGPT are highly aligned with known cell labels and states. The feature patterns of tumor bulk tissues learned by tGPT are associated with a wide range of genomic alteration events, prognosis, and treatment outcome of immunotherapy. tGPT represents a new analytical paradigm for integrating and deciphering massive amounts of transcriptome data and it will facilitate the interpretation and clinical translation of single-cell transcriptomes.

Pancreatic cancer is difficult to diagnose at the initial stage and is often discovered after metastasis to nearby organs. Gemcitabine is currently used as a standard treatment for pancreatic cancer. However, since chemotherapy for pancreatic cancer has not yet reached satisfactory therapeutic results, adjuvant chemotherapy methods are attempted. It can be expected that combining immune cell therapy with existing anticancer drug combination treatment will prevent cancer recurrence and increase survival rates. We isolated natural killer (NK) cells and co-cultured them with strongly activated autologous peripheral blood mononuclear cells (PBMCs) as feeder cells, activated using CD3 antibody, IFN-r, IL-2, and γ-radiation. NK cells expanded in this method showed greater cytotoxicity than resting NK cells, when co-cultured with pancreatic cancer cell lines. Tumor growth was effectively inhibited in a pancreatic cancer mouse xenograft model. Therapeutic efficacy was increased by using gemcitabine and erlotinib in combination. These findings suggest that NK cells cultured by the method proposed here have excellent anti-tumor activity. We demonstrate that activated NK cells can efficiently inhibit pancreatic tumors when used in combination with gemcitabine-based therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is critical for natural killer (NK) cell-mediated anti-tumor and anti-microbe killing. The TRAIL expression on the donor's liver NK cells from the liver perfusate after interleukin-2 stimulation varies between individuals and is unpredictable. This study aimed to clarify the risk factors for low TRAIL expression by analyzing perioperative donor characteristics.

This retrospective study of living donor liver transplant (LDLT) donors between 2006 and 2022 was performed to analyze low TRAIL expression risk factors. Seventy-five donors who had undergone hepatectomy for LDLT were divided into 2 groups, low and high TRAIL, according to their TRAIL expression on liver NK cells, using median values.

The low TRAIL group (N = 38) was older and had lower nutrition and a higher low-density lipoprotein/high-density lipoprotein (LDL/HDL) cholesterol ratio, related to arteriosclerosis, than the high TRAIL group (N = 37). In multivariate analysis, the geriatric nutritional risk index (GNRI) (odds ratio, 0.86; 95% CI, 0.76-0.94; P < .001) and LDL/HDL cholesterol ratio (odds ratio, 2.32; 95% CI, 1.10-4.86; P = .005) were independent predictive factors for low TRAIL expression on liver NK cells. Furthermore, the TRAIL expression of liver NK cells decreased in donors who already had atherosclerosis and in donors at risk of potentially developing atherosclerosis.

The TRAIL expression on liver NK cells in donors had a strong relationship with atherosclerosis and GNRI. Atherosclerosis can reflect the TRAIL expression on liver NK cells.

Two extracellular domains of the adhesive receptor DNAM-1 are involved in various cellular biological processes through binding to ligand CD155, usually under a mechano-microenvironment. The first extracellular domain (D1) plays a key role in recognition, but the function of the second extracellular domain (D2) and effects of force on the interaction of DNAM-1 with CD155 remain unclear. We herein studied the interaction of DNAM-1 with CD155 by performing steered molecular dynamics (MD) simulations, and observed the roles of tensile force and D2 on the affinity of DNAM-1 to CD155. The results showed that D2 improved DNAM-1 affinity to CD155; the DNAM-1/CD155 complex had a high mechanical strength and a better mechanical stability for its conformational conservation either at pulling with constant velocity or under constant tensile force (≤100 pN); the catch-slip bond transition governed CD155 dissociation from DNAM-1; and, together with the newly assigned key residues in the binding site, force-induced conformation changes should be responsible for the mechanical regulation of DNAM-1's affinity to CD155. This work provided a novel insight in understanding the mechanical regulation mechanism and D2 function in the interaction of DNAM-1 with CD155, as well as their molecular basis, relevant transmembrane signaling, and cellular immune responses under a mechano-microenvironment.

Using a comprehensive flow cytometric panel, simultaneously obtained mid-luteal immunophenotypes from peripheral blood and endometrium were compared and values correlated. Is a peripheral blood evaluation of reproductive immunophenotype status meritorious relative to local endometrial evaluation to directly assess the peri-implantation environment?

Fifty-five patients had a mid-luteal biopsy to assess the local endometrial immunophenotype, while simultaneously providing a peripheral blood sample for analysis. Both samples were immediately assessed using a comprehensive multi-parameter panel, and lymphocyte subpopulations were described and compared.

Distinct lymphocyte proportions and percentage differences were noted across the two compartments, confirming the hypothesis that they are distinct environments. The ratio of CD4 + to CD8 + T cells were reversed between the two compartments, as were Th1 and Th2-type CD4 + T cell ratios. Despite these differences, some direct relationships were noted. Positive Pearson correlations were found between the levels of CD57 + expressing natural killer cells, CD3 + NK-T cells and CD4 + Th1 cells in both compartments.

Flow cytometric evaluation provides a rapid and objective analysis of lymphocyte subpopulations. Endometrial biopsies have become the gold standard technique to assess the uterine immunophenotype in adverse reproductive outcome, but there may still a place for peripheral blood evaluation in this context. The findings demonstrate significant variations in cellular proportions across the two regions, but some positive correlations are present. Immunological assessment of these specific peripheral blood lymphocyte subtypes may provide insight into patients with potential alterations of the uterine immune environment, without the risks and inconveniences associated with an invasive procedure.

Type 1 diabetes (T1D) is a multifactorial autoimmune disease, involving strong genetic components with familial predisposition. Killer cell immunoglobulin-like receptors (KIRs) found on the surface of NK cells have ligands of human leukocyte antigen (HLA) class I that are associated with T1D. The present study evaluates the influence of KIR genes and their HLA-ligands in the aetiology of T1D among the South Indian population.

A total of 125 T1D patients, along with their parents (n = 126) and siblings (n = 52) were recruited. PCR-based genotyping was performed for KIR genes and HLA class I ligands. The gene frequencies were compared between patients and siblings/parents. Linkage-disequilibrium (LD) analysis was performed to assess the genetic association between KIR gene pairs.

The results show significant differences in HLA-ligands of KIR genes between patients and parents. The HLA-C1C1 homozygosity was found to be a predisposing risk factor, and HLA-C1C2 heterozygosity was protective towards T1D along with either the activating KIR2DS2 or inhibitory KIRs 2DL1, 2DL2, 2DL3. However, the frequency of inhibitory KIR3DL1 significantly increased in the presence of HLA-B Bw4 Ile80 in parents when compared to patients showing a protective effect on T1D. Two pairs of KIR genes, 2DS4-3DL1 and 2DS1-2DL5, showed strong LD in patients, siblings and parents.

The KIR-HLA ligand combinations have a significant effect on T1D aetiology among the South Indian population. This study defines a pattern for family-based association studies with genotypic information about KIR genes and their HLA-ligands, providing the first evidence towards T1D among the South Indian population.

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV⁺ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV⁺PLWH and CMV⁺HIV^- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV⁺PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons.

Lactobacillus paragasseri SBT2055 (LG2055) has been reported to show immunostimulating effects. This study aimed to investigate the effects of LG2055 on the subjective symptoms of the physical condition in healthy adults.

In this randomized, double-blind, placebo-controlled, parallel-group comparative study, Japanese individuals aged 20-64 years were recruited. A total of 200 participants were randomly divided into two groups by an independent controller (LG2055 and placebo groups; 100 participants per group). Drinkable yogurts containing LG2055 or lacking LG2055 (placebo) were used as test samples. The participants ingested one bottle of the test sample once a day for 12 weeks. A daily physical health questionnaire survey (about common cold symptoms) was performed as the primary outcome, and immunological and oxidative stress markers in saliva and serum were evaluated as secondary outcomes.

In total, 198 participants completed the scheduled intake of the test samples, and five participants were excluded from the final analysis. Consequently, 193 participants (LG2055 group, n = 97; placebo group, n = 96) in the Per-Protocol Set were included in the efficacy analysis. The cumulative days of each symptom were evaluated, and the LG2055 group showed a significantly higher ratio of "without symptom" in runny nose, plugged nose, sneezing, sore throat, hoarseness, cough, headache, feeling tired, and fever than the placebo group, indicating that the incidence rates of common cold symptoms were lower in the LG2055 group. Additionally, changes in the salivary secretory IgA levels were significantly higher, and the serum derivatives of reactive oxygen metabolites levels were significantly lower in the LG2055 group.

Our study revealed that intake of LG2055 decreased common cold symptoms and improved immune parameters in healthy adults. This suggests that LG2055 contributes to the maintenance of physical conditions by improving the host immune system.

[https://www.umin.ac.jp/ctr/index.htm], identifier [UMIN000045901].