Regulate Natural Killer Cell Research Articles

Combination Effect of Radiotherapy and Targeted Therapy with NK Cell-Based Immunotherapy in head and Neck Squamous Cell Carcinoma

ABSTRACT Background Head and neck squamous cell carcinoma (HNSCC) has a poor prognosis, and current treatments are limited by high toxicity and low survival rates, highlighting the need for new therapeutic approaches. Natural killer (NK) cells can identify and eliminate cancer cells without prior antigen exposure. Radiotherapy directly targets tumors and increases activating ligands on tumor cells, promoting NK cell interactions. Cetuximab, an EGFR-targeting antibody, enhances NK cell cytotoxicity. Additionally, anti-PD-1 antibodies may further boost NK cell function by blocking inhibitory signals. The study aimed to enhance HNSCC treatment efficacy by combining radiotherapy and targeted therapy with expanded NK cells. Methods NK cells were isolated, activated, and expanded from healthy donors. The FaDu and SCC-47 cell lines were inoculated into NOD/SCID mice. The mice were treated with PD-1 inhibitors, cetuximab, and radiation, followed by intravenous injection of NK cells. Results Radiation increased ligands that regulate NK cell sensitivity. The combination of cetuximab, radiotherapy, and expanded NK cells significantly suppressed cancer progression and improved survival rates. However, adding anti-PD-1 antibodies did not further enhance outcomes. Conclusion This study suggests that a multimodal approach combining cetuximab, radiotherapy, and NK cells can significantly improve HNSCC therapy efficacy, offering a novel and promising treatment strategy.

Protein phosphatase 2A inhibitor modulates natural killer cell homeostasis in peripheral tissues

Although natural killer (NK) cell responses to tumor and viral infection have been studied, the mechanisms underlying NK cell homeostasis in vivo remain unclear. In this study, we demonstrate the pharmacological action of cytostatin, a protein phosphatase 2A (PP2A) specific inhibitor (PP2Ai), on NK cells in regulating NK cell homeostasis in the peripheral tissues. We found that PP2Ai treatment decreased NK cell percentages in the bone marrow and secondary lymphoid tissues while increasing NK cell percentages in peripheral tissues such as the lung and liver. In the peripheral tissues of PP2Ai-treated mice, Ki-67 expression and BrdU uptake in NK cells were upregulated, and an initial increase in the pre-mature CD11bhiCD27hi NK subset was observed, followed by an increase in the terminally differentiated mature CD11bhiCD27lo NK subset. In addition, bone marrow Ki-67+ NK cells predominantly expressed CX3CR1 in the PP2Ai-treated mice and were further mobilized to the peripheral tissues. Among various target molecules of PP2A, we found that the upregulation of c-Myc pathway and its phosphorylation, along with its downstream cyclin E expression and G1/S cell cycle transition in PP2Ai-treated mice NK cells. Our results suggest that PP2Ai modulates NK cell proliferation through c-Myc and cyclin E, leading to their maturation and trafficking from the bone marrow to the peripheral tissues.

Arachidonic acid impairs natural killer cell functions by disrupting signaling pathways driven by activating receptors and reactive oxygen species

BackgroundHigh levels of the polyunsaturated fatty acid arachidonic acid (AA) within the ovarian carcinoma (OC) microenvironment correlate with reduced relapse-free survival. Furthermore, OC progression is tied to compromised immunosurveillance, partially attributed to the impairment of natural killer (NK) cells. However, potential connections between AA and NK cell dysfunction in OC have not been studied.MethodsWe employed a combination of phosphoproteomics, transcriptional profiling and biological assays to investigate AA’s impact on NK cell functions.ResultsAA (i) disrupts interleukin-2/15-mediated expression of pro-inflammatory genes by inhibiting STAT1-dependent signaling, (ii) hampers signaling by cytotoxicity receptors through disruption of their surface expression, (iii) diminishes phosphorylation of NKG2D-induced protein kinases, including ERK1/2, LYN, MSK1/2 and STAT1, and (iv) alters reactive oxygen species production by transcriptionally upregulating detoxification. These modifications lead to a cessation of NK cell proliferation and a reduction in cytotoxicity.ConclusionOur findings highlight significant AA-induced alterations in the signaling network that regulates NK cell activity. As low expression of several NK cell receptors correlates with shorter OC patient survival, these findings suggest a functional linkage between AA, NK cell dysfunction and OC progression.

Dichotomous outcomes of TNFR1 and TNFR2 signaling in NK cell-mediated immune responses during inflammation

Natural killer (NK) cell function is regulated by a balance of activating and inhibitory signals. Tumor necrosis factor (TNF) is an inflammatory cytokine ubiquitous across homeostasis and disease, yet its role in regulation of NK cells remains unclear. Here, we find upregulation of the immune checkpoint protein, T cell immunoglobulin and mucin domain 3 (Tim3), is a biomarker of TNF signaling in NK cells during Salmonella Typhimurium infection. In mice with conditional deficiency of either TNF receptor 1 (TNFR1) or TNF receptor 2 (TNFR2) in NK cells, we find TNFR1 limits bacterial clearance whereas TNFR2 promotes it. Mechanistically, via single cell RNA sequencing we find that both TNFR1 and TNFR2 induce the upregulation of Tim3, while TNFR1 accelerates NK cell death but TNFR2 promotes NK cell accumulation and effector function. Our study thus highlights the complex interplay of TNF-based regulation of NK cells by the two TNF receptors during inflammation.

Emerging Role of the p53 Pathway in Modulating Natural Killer Cell Mediated Immunity.

The p53 pathway plays an important role in role in cancer immunity. Mutation or downregulation of the proteins in the p53 pathway are prevalent in many cancers, contributing to tumor progression and immune dysregulation. Recent findings suggest that the activity of p53 within tumor cells, immune cells, and the tumor microenvironment can play an important role modulating natural killer (NK) cell-mediated immunity. Consequently, efforts to restore p53 pathway activity are being actively pursued to modulate this form of immunity. This review focuses on p53 activity regulating the infiltration and the activation of NK cells in the tumor immune microenvironment, which are illustrated in the Graphical Abstract for this review. Furthermore, impact of p53 and its regulation of NK cells on immunogenic cell death within solid tumors and the abscopal effect is reviewed. Finally, future avenues for therapeutically restoring p53 activity to improve NK cell-mediated anti-tumor immunity and optimize the effectiveness of cancer therapies are discussed.

Interferon-mediated NK cell activation is associated with limited neutralization breadth during SARS-CoV-2 infection.

Best known for their ability to kill infected or malignant cells, natural killer (NK) cells are also underappreciated regulators of the antibody response to viral infection. In mice, NK cells can kill T follicular helper (Tfh) cells, decreasing somatic hypermutation and vaccine responses. Although human NK cell activation correlates with poor vaccine response, the mechanisms of human NK cell regulation of adaptive immunity are poorly understood. We found that in human ancestral SARS-CoV-2 infection, broad neutralizers, who were capable of neutralizing Alpha, Beta, and Delta, had fewer NK cells that expressed inhibitory and immaturity markers whereas NK cells from narrow neutralizers were highly activated and expressed interferon-stimulated genes (ISGs). ISG-mediated activation in NK cells from healthy donors increased cytotoxicity and functional responses to induced Tfh-like cells. This work reveals that NK cell activation and dysregulated inflammation may play a role in poor antibody response to SARS-CoV-2 and opens exciting avenues for designing improved vaccines and adjuvants to target emerging pathogens.

DNMT1 silencing induces KIR2DL1/2/3 expression via methylation to alleviate graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Natural killer (NK) cells are the promoters in graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), while demethylation can regulate NK cell function. We explored the mechanism of demethylation regulating NK cell function to affect GVHD after allo-HSCT. BALB/c mice were transfused with C57BL/6 mouse-derived NK and bone marrow cells to establish GVHD models, followed by isolation and in-vitro expansion of NK cells. NK cell purity, cytokine levels, proliferation, and cytokine-producing NK cell levels were measured via flow cytometry. KIR2DL1/2/3 methylation was tested by Methylation-specific polymerase chain reaction (MSP), with determination of mouse survival and GVHD scores. KIR2DL1/2/3 and DNMT1 expression was detected through qRT-PCR and/or western blot. Methylation levels were upregulated and KIR2DL1/2/3 expression was downregulated in GVHD mouse model-derived NK cells following IL-2 stimulation. DNMT1 silencing promoted KIR2DL1/2/3 expression, proliferation, and the secretion of Granzyme, Perforin, and Interferon-γ (IFN-γ) in C57BL/6 mouse-derived NK cells. DNMT1 silencing also enhanced mouse survival, reduced GVHD scores, promoted KIR2DL1/2/3 expression on the NK cell surface, and increased the secretion of Granzyme, Perforin, IFN-γ, and the number of cytokine-producing NK cells in the spleen, liver, and lung tissues of the models. Collectively, DNMT1 silencing induced KIR2DL1/2/3 expression in NK cells through reducing methylation to alleviate GVHD after allo-HSCT.

Surface Immune Checkpoints as Potential Biomarkers in Physiological Pregnancy and Recurrent Pregnancy Loss.

Due to the genetic diversity between the mother and the fetus, heightened control over the immune system during pregnancy is crucial. Immunological parameters determined by clinicians in women with idiopathic recurrent spontaneous abortion (RSA) include the quantity and activity of Natural Killer (NK) and Natural Killer T (NKT) cells, the quantity of regulatory T lymphocytes, and the ratio of pro-inflammatory cytokines, which indicate imbalances in Th1 and Th2 cell response. The processes are controlled by immune checkpoint proteins (ICPs) expressed on the surface of immune cells. We aim to investigate differences in the expression of ICPs on T cells, T regulatory lymphocytes, NK cells, and NKT cells in peripheral blood samples collected from RSA women, pregnant women, and healthy multiparous women. We aim to discover new insights into the role of ICPs involved in recurrent pregnancy loss. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from blood samples obtained from 10 multiparous women, 20 pregnant women (11-14th week of pregnancy), and 20 RSA women, at maximum of 72 h after miscarriage. The PBMCs were stained for flow cytometry analysis. Standard flow cytometry immunophenotyping of PBMCs was performed using antibodies against classical lymphocyte markers, including CD3, CD4, CD8, CD56, CD25, and CD127. Additionally, ICPs were investigated using antibodies against Programmed Death Protein-1 (PD-1, CD279), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3, CD366), V-domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), and Lymphocyte activation gene 3 (LAG-3). We observed differences in the surface expression of ICPs in the analyzed subpopulations of lymphocytes between early pregnancy and RSA, after miscarriage, and in women. We noted diminished expression of PD-1 on T lymphocytes (p = 0.0046), T helper cells (CD3CD4 positive cells, p = 0.0165), T cytotoxic cells (CD3CD8 positive cells, p = 0.0046), T regulatory lymphocytes (CD3CD4CD25CD127 low positive cells, p = 0.0106), and NKT cells (CD3CD56/CD16 positive cells, p = 0.0438), as well as LAG-3 on lymphocytes T (p = 0.0225) T helper, p = 0.0426), T cytotoxic cells (p = 0.0458) and Treg (p = 0.0293), and cells from RSA women. Impaired expression of TIM-3 (p = 0.0226) and VISTA (p = 0.0039) on CD8 cytotoxic T and NK (TIM3 p = 0.0482; VISTA p = 0.0118) cells was shown, with an accompanying increased expression of TIGIT (p = 0.0211) on NKT cells. The changes in the expression of surface immune checkpoints indicate their involvement in the regulation of pregnancy. The data might be utilized to develop specific therapies for RSA women based on the modulation of ICP expression.

Natural Killer Cell Regulation of Breast Cancer Stem Cells Mediates Metastatic Dormancy.

Patients with breast cancer with estrogen receptor-positive tumors face a constant risk of disease recurrence for the remainder of their lives. Dormant tumor cells residing in tissues such as the bone marrow may generate clinically significant metastases many years after initial diagnosis. Previous studies suggest that dormant cancer cells display "stem-like" properties (cancer stem cell, CSC), which may be regulated by the immune system. To elucidate the role of the immune system in controlling dormancy and its escape, we studied dormancy in immunocompetent, syngeneic mouse breast cancer models. Three mouse breast cancer cell lines, PyMT, Met1, and D2.0R, contained CSCs that displayed short- and long-term metastatic dormancy in vivo, which was dependent on the host immune system. Each model was regulated by different components of the immune system. Natural killer (NK) cells were key for the metastatic dormancy phenotype in D2.0R cells. Quiescent D2.0R CSCs were resistant to NK cell cytotoxicity, whereas proliferative CSCs were sensitive. Resistance to NK cell cytotoxicity was mediated, in part, by the expression of BACH1 and SOX2 transcription factors. Expression of STING and STING targets was decreased in quiescent CSCs, and the STING agonist MSA-2 enhanced NK cell killing. Collectively, these findings demonstrate the role of immune regulation of breast tumor dormancy and highlight the importance of utilizing immunocompetent models to study this phenomenon. Significance: The immune system controls disseminated breast cancer cells during disease latency, highlighting the need to utilize immunocompetent models to identify strategies for targeting dormant cancer cells and reducing metastatic recurrence. See related commentary by Cackowski and Korkaya, p. 3319.

Proteomic analysis of granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex by MALDI imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

Natural killer cells associate with epithelial cells in the pancreatic ductal adenocarcinoma tumor microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. PDAC's poor prognosis and resistance to immunotherapy are attributed in part to its dense, fibrotic tumor microenvironment (TME), which is known to inhibit immune cell infiltration. We recently demonstrated that PDAC patients with higher natural killer (NK) cell content and activation have better survival rates. However, NK cell interactions in the PDAC TME have yet to be deeply studied. We show here that NK cells are present and active in the human PDAC TME. We used imaging mass cytometry (IMC) to assess NK cell content, function, and spatial localization in human PDAC samples. Then, we used CellChat, a tool to infer ligand-receptor interactions, on a human PDAC scRNAseq dataset to further define NK cell interactions in PDAC. Spatial analyses showed for the first time that active NK cells are present in the PDAC TME, and both associate and interact with malignant epithelial cell ducts. We also found that fibroblast-rich, desmoplastic regions limit NK cell infiltration in the PDAC TME. CellChat analysis identified that the CD44 receptor on NK cells interacts with PDAC extracellular matrix (ECM) components such as collagen, fibronectin and laminin expressed by fibroblasts and malignant epithelial cells. This led us to hypothesize that these interactions play roles in regulating NK cell motility in desmoplastic PDAC TMEs. Using 2D and 3D in vitro assays, we found that CD44 neutralization significantly increased NK cell invasion through matrix. Targeting ECM-immune cell interactions may increase NK cell invasion into the PDAC TME.

Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.

Contribution of Helios in Ets1-Deficient NK Cells

Abstract Natural killer (NK) cells mediate anti-viral and anti-cancer immune responses. However, the gene networks that regulate NK cell development and function remain poorly understood. One transcription factor critical for proper NK cell development is Ets1. Previously we showed that loss of Ets1 at the germline level resulted in a developmental block, a functional defect, as well as a phenotype reminiscent of chronic cytokine stimulation in the NK cell compartment. Ets1-deficient NK cells also overexpress Helios, an Ikaros family transcription factor encoded by Ikzf2. Ikaros family transcription factors regulate development in many hematopoietic cell types; therefore, we hypothesized that Helios may be acting downstream of Ets1 to mediate the observed developmental block or functional defects. To investigate this, we generated NK cell-driven conditional deletion of Ets1 only (Ets1 cKO) or Ets1 and Ikzf2 (2xKO). The data presented here confirmed that Helios is highly expressed at the protein level in Ets1 cKO NK cells. Further, overexpression of Helios in Ets1-deficient NK cells does not contribute to the block in maturation nor the cytokine production defect. However, activated NK cells show high Helios expression, suggesting that Helios expression in Ets1-/- NK cells may be a consequence of the chronic cytokine stimulation phenotype.

Vitamin A-treated natural killer cells reduce interferon-gamma production and support regulatory T-cell differentiation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that contribute to immune responses against stressed, transformed, or infected cells. NK cell effector functions are regulated by microenvironmental factors, including cytokines, metabolites, and nutrients. Vitamin A is an essential micronutrient that plays an indispensable role in embryogenesis and development, but was also reported to regulate immune responses. However, the role of vitamin A in regulating NK cell functions remains poorly understood. Here, we show that the most prevalent vitamin A metabolite, all-trans retinoic acid (atRA), induces transcriptional and functional changes in NK cells leading to altered metabolism and reduced IFN-γ production in response to a wide range of stimuli. atRA-exposed NK cells display a reduced ability to support dendritic cell (DC) maturation and to eliminate immature DCs. Moreover, they support the polarization and proliferation of regulatory T cells. These results imply that in vitamin A-enriched environments, NK cells can acquire functions that might promote tolerogenic immunity and/or immunosuppression.

MEF2C regulates NK cell effector functions through control of lipid metabolism.

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.

Natural Killer Cell Subsets in Tumor Draining Lymph Nodes of Patients with Bladder Cancer and Their Clinical Implications.

Natural killer (NK) cells are crucial innate components in anti-tumor immunity. However, the clinical impacts and their phenotypes in bladder cancer (BC) remain unclear. To assess the clinical significance of NK cell subsets in tumor-draining lymph nodes of patients with BC. In a cross-sectional study, pelvic lymph nodes were obtained from 49 untreated patients with BC. Mononuclear cells were isolated and immunophenotyped using CD3, CD56, CD16, CD27, and CD11b markers. NK cells were then classified based on their expression patterns of CD56/CD16 (conventional) and CD27/CD11b (new). On average, NK cells constituted 2.99±1.44% of the total lymphocytes in the draining lymph node of patients with BC. The CD56dim and regulatory NK subsets (CD27+CD11b+/-) were the predominant old and new NK, respectively. The NK cells significantly increased in patients with at least one involved node (LN+) compared with those with free nodes (LN-; p=0.022). Conversely, CD56dimCD16- subset significantly decreased in higher stages (p=0.032) and in tumors with muscle invasion (p=0.038). Significant variations were also observed in different T-stages (p<0.05). Regarding new classification, the frequency of CD11b+ regulatory NK cells was significantly lower in node-positive patients (p=0.025). These findings emphasize the dynamic nature of NK cell subsets in bladder cancer and their potential relevance in disease progression and management, suggesting potential implications for therapeutic strategies targeting these specific subsets.

Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

Impact of KIR-HLA Genotype on Natural-Killer-Cell-Based Immunotherapy for Preventing Hepatocellular Carcinoma after Living-Donor Liver Transplantation

Natural killer (NK) cells have immunosurveillance potential in hepatocellular carcinoma (HCC). We performed adaptive immunotherapy using donor-liver-derived natural killer (NK) cells after living-donor liver transplantation (LDLT) to prevent HCC recurrence. Dominant inhibitory signals tightly regulate NK cell activity via human leukocyte antigen (HLA)-specific inhibitory receptors, such as killer immunoglobulin-like receptors (KIRs). The functional recognition of HLA through KIR raises the NK cell capacity, which is a process termed “licensing.” Here, we investigated the effect of polymorphic KIR-HLA genotypes on the efficacy of NK-cell-based immunotherapy after LDLT. Seventy-seven Japanese recipients with HCC who underwent LDLT and their corresponding donors between 1996 and 2016 were enrolled in this study. The median follow-up period was 8.3 years. The HCC recurrence risk was stratified using radiological and pathological assessments according to the Milan criteria. Of the 77 recipients, 38 received immunotherapy. Immunotherapy improves early post-transplantation survival and lowers the recurrence rate in the intermediate-risk recipients. We analyzed the genotypes of five inhibitory KIRs and HLA using sequence-specific polymorphism-based typing. The polymorphic KIR-HLA genotype revealed that genetically vulnerable liver transplant recipients with a poorly licensed NK genotype have an improved prognosis by immunotherapy with donor-liver-derived NK cells. Thus, the combination of recipient and donor KIR-HLA genotypes is worthy of attention for further investigation, especially considering the clinical application of NK-cell-based immunotherapy.

Interactions between Immunoglobulin like receptors and the Peptidome in the Allogeneic Hematopoietic Stem Cell Transplantation in the Cytomegalovirus context. A mini review.

The Natural Killer cells are innate lymphoid cells that play essential roles in defense against viral and parasitic infections, elimination of tumor cells, regulation of adaptive immunity through cytotoxicity, and cytokine secretion. Fundamental knowledge about the regulation of Natural Killer cells can be applied to study their function in patients undergoing hematopoietic stem cell transplantation, with or without Cytomegalovirus reactivation. The function of Natural Killer cells is governed by a repertoire of receptors responsible for initiating intracellular activating or inhibitory signaling. The balance of this signaling directs the cytotoxic activity of these cells, as well as cell proliferation and cytokine release.Understanding the interaction of receptors expressed on the surface of Natural Killer cells with their ligands expressed on target cells is a topic of discussion in the context of alloreactivity and the graft-versus-leukemia effect in transplant patients. Recent investigations have shown that KIR/HLA interactions go beyond affinity and describe that many of them depend on the peptide being presented by the HLA at that moment. Analysis of the peptidome (HLA class I + bound peptide) has demonstrated that some Natural Killer cell receptors are peptide-dependent. Therefore, understanding these interactions by considering the entire Natural Killer cell receptor + HLA class I + peptide complex is crucial in the reestablishment of immune cells after hematopoietic stem cell transplantation, especially in the context of Cytomegalovirus reactivation, which is very common in these patients. Hence, the aim of this study is to deepen our understanding of the specificity of interactions between human Natural Killer cell KIR receptors and the peptidome in the context of Cytomegalovirus reactivation after allogeneic, related, HLA-compatible hematopoietic stem cell transplantation without T-cell depletion. For the analysis of interactions between Natural Killer cell receptors and peptidomes, specific peptide libraries for HLA class I alleles will be created to evaluate the specificities of interactions between KIR + HLA class I + peptide.