ABSTRACT
Chimeric antigen receptor-transduced autologous T (CAR-T) cell therapy targeting CD19 has revolutionized the treatment of CD19-positive hematological tumors, including acute lymphoblastic leukemia and large B-cell lymphoma. However, despite the high response rate, many problems such as exceedingly high cost, complex logistics, insufficient speed, and manufacturing failures have become apparent. One solution for these problems is to use an allogeneic cell as an effector cell for genetic modification with CAR. Allogeneic, or "off-the-shelf", CAR-expressing immune-effector cells include 1) genome-edited, T-cell receptor (TCR) gene-deleted CAR-T cells generated using healthy adult donor T cells, 2) induced pluripotent stem cell-derived CAR-T cells, and 3) CAR NK cells. NK cells are notorious for their poor ex-vivo expansion and low susceptibility to genetic modification. In this article, I will review the current state and future prospects of allogeneic CAR cell therapies, with special reference to CAR NK cells.
Subject(s)
Killer Cells, Natural , Humans , Killer Cells, Natural/immunology , Transplantation, Homologous , Receptors, Chimeric Antigen/immunology , Immunotherapy, Adoptive/methodsABSTRACT
Hematopoietic cell transplantation (HCT) represents a potentially curative therapeutic approach for various hematologic and non-hematologic malignancies. Human leukocyte antigen (HLA) matching is still the central selection criterion for HCT donors. Nevertheless, post-transplant complications, in particular graft-versus-host disease (GvHD), relapse of disease and infectious complications, represent a major challenge and contribute significantly to morbidity and mortality. Recently, non-classical HLA class I molecules, especially HLA-E, have gained increasing attention in the context of allogeneic HCT. This review aims to summarize the latest findings on the immunomodulatory role of HLA-E, which serves as a ligand for receptors of the innate and adaptive immune system. In particular, we aim to elucidate how (i) polymorphisms within HLA-E, (ii) the NKG2A/C axis and (iii) the repertoire of peptides presented by HLA-E jointly influence the functionality of immune effector cells. Understanding this intricate network of interactions is crucial as it significantly affects NK and T cell responses and thus clinical outcomes after HCT.
Subject(s)
HLA-E Antigens , Hematopoietic Stem Cell Transplantation , Histocompatibility Antigens Class I , Killer Cells, Natural , Humans , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/genetics , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily C/immunology , NK Cell Lectin-Like Receptor Subfamily C/genetics , Graft vs Host Disease/immunology , Graft vs Host Disease/genetics , Allografts , T-Lymphocytes/immunology , Polymorphism, Genetic , Transplantation, HomologousABSTRACT
Skin cancer has the highest incidence of all cancers, and their incidence are increasing in both melanoma and non-melanoma skin cancers. Alternative adjuvant treatment strategies appropriate for their management are needed. Modifiable lifestyle factors influence disease outcomes, either improving or worsening outcomes. Exercise is an example of a modifiable lifestyle factor, and can be prescribed as an adjuvant therapy in other cancer types to improve immune function and overall clinical outcomes. The initial aim of the review was to investigate the T-cell specific mechanisms of exercise which affect clinical/disease outcomes in skin cancer. Study quality was assessed by a modified Covidence quality assessment template with animal-model study specific criteria. A total of 10 articles were included; all articles were murine model studies investigating melanoma. Eight studies (n=8) employed a randomised controlled trial design, with two bio-informatics studies, and one study using human data which could solidify a link to human health. While the review focussed initially on T-cells, many studies reported significant changes in NK cells, and as they share the same haematopoietic lineage/ common lymphoid progenitor as T cells, the data was included in the analyses. Most studies indicated that exercise reduced melanoma tumour burden. Exercising prior to melanoma inoculation was most effective for delaying carcinogenesis and reducing tumour burden. Synergism was a topic identified in studies; PD-1/PD-L1 treatment, and exercise were not synergistic. Conversely, exercise and mental stimulation were synergistic, and the temperature at which exercise was conducted significantly reduced tumour burden. Several murine studies reported that exercise improved clinical outcomes in melanoma, and that long-term exercise was more effective in reducing tumour burden. Further studies are required to investigate this relationship in humans, and in other types of skin cancer.
Subject(s)
Exercise , Killer Cells, Natural , Skin Neoplasms , T-Lymphocytes , Skin Neoplasms/immunology , Skin Neoplasms/therapy , Humans , Animals , Killer Cells, Natural/immunology , Exercise/physiology , T-Lymphocytes/immunology , Melanoma/immunology , Melanoma/therapy , Mice , Disease Models, AnimalABSTRACT
NK cells are cytotoxic innate immune cells involved in antitumor immunity, and they provide a treatment option for patients with acute myeloid leukemia (AML). In this issue of the JCI, Cubitt et al. investigated the role of CD8α, a coreceptor present on approximately 40% of human NK cells. IL-15 stimulation of CD8α- NK cells induced CD8α expression via the RUNX3 transcription factor, driving formation of a unique induced CD8α (iCD8α+) population. iCD8α+ NK cells displayed higher proliferation, metabolic activity, and antitumor cytotoxic function compared with preexisting CD8α+ and CD8α- subsets. Therefore, CD8α expression can be used to define a potential dynamic spectrum of NK cell expansion and function. Because these cells exhibit enhanced tumor control, they may be used to improve in NK cell therapies for patients with AML.
Subject(s)
CD8 Antigens , Core Binding Factor Alpha 3 Subunit , Interleukin-15 , Killer Cells, Natural , Leukemia, Myeloid, Acute , Humans , CD8 Antigens/metabolism , CD8 Antigens/immunology , CD8 Antigens/genetics , Core Binding Factor Alpha 3 Subunit/genetics , Core Binding Factor Alpha 3 Subunit/metabolism , Core Binding Factor Alpha 3 Subunit/immunology , Interleukin-15/immunology , Interleukin-15/metabolism , Interleukin-15/genetics , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolismABSTRACT
Introduction: Heme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation, and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors, and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation, that is, NOD-like receptor protein 3 (NLRP3), glutathione (GSH), GSH disulfide (GSSG), and interleukin 6 (IL-6). Methods: The study population comprised three age groups: young adults (age range, 19-23 years), older adults aged under 85 years (age range, 73-84 years), and older adults aged over 85 years (age range, 85-92 years). NLRP3, GSH, and GSSG concentrations were measured in serum, whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2, lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate (PMA) with ionomycin. Results: The analysis of serum NLRP3, GSH, and GSSG concentrations revealed no statistically significant differences among the studied age groups. However, some typical trends of aging were observed, such as a decrease in GSH concentration and an increase in both GSSG level, and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover, statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells. Conclusions: These results showed that NK cells can express HO-1 at a basal level, which was significantly increased in activated cells, even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters.
Subject(s)
Aging , Heme Oxygenase-1 , Killer Cells, Natural , Oxidative Stress , Humans , Heme Oxygenase-1/metabolism , Aging/immunology , Aged, 80 and over , Aged , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Young Adult , Female , Glutathione/metabolism , Interleukin-6/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , AdultABSTRACT
Introduction: NK cells can mediate tumor cell killing by natural cytotoxicity and by antibody-dependent cell-mediated cytotoxicity (ADCC), an anti-tumor mechanism mediated through the IgG Fc receptor CD16A (FcγRIIIA). CD16A polymorphisms conferring increased affinity for IgG positively correlate with clinical outcomes during monoclonal antibody therapy for lymphoma, linking increased binding affinity with increased therapeutic potential via ADCC. We have previously reported on the FcγR fusion CD64/16A consisting of the extracellular region of CD64 (FcγRI), a high-affinity Fc receptor normally expressed by myeloid cells, and the transmembrane/cytoplasmic regions of CD16A, to create a highly potent and novel activating fusion receptor. Here, we evaluate the therapeutic potential of engineered induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as an "off-the-shelf", antibody-armed cellular therapy product with multi-antigen targeting potential. Methods: iNK cells were generated from iPSCs engineered to express CD64/16A and an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein for cytokine independence. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells to examine in in vitro and in vivo assays using the Raji lymphoma cell line. ADCC was evaluated in real-time by IncuCyte assays and using a xenograft mouse model with high circulating levels of human IgG. Results: Our data show that CD64/16A expressing iNK cells can mediate potent anti-tumor activity against human B cell lymphoma. In particular, (i) under suboptimal conditions, including low antibody concentrations and low effector-to-target ratios, iNK-CD64/16A cells mediate ADCC, (ii) iNK-CD64/16A cells can be pre-loaded with tumor-targeting antibodies (arming) to elicit ADCC, (iii) armed iNK-CD64/16A cells can be repurposed with additional antibodies to target new tumor antigens, and (iv) cryopreserved, armed iNK-CD64/16A are capable of sustained ADCC in a tumor xenograft model under saturating levels of human IgG. Discussion: iNK-CD64/16A cells allow for a flexible use of antibodies (antibody arming and antibody targeting), and an "off-the-shelf" platform for multi-antigen recognition to overcome limitations of adoptive cell therapies expressing fixed antigen receptors leading to cancer relapse due to antigen escape variants.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , Antigens, Neoplasm , Induced Pluripotent Stem Cells , Killer Cells, Natural , Lymphoma , Receptors, IgG , Xenograft Model Antitumor Assays , Receptors, IgG/immunology , Receptors, IgG/metabolism , Receptors, IgG/genetics , Humans , Animals , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Mice , Lymphoma/therapy , Lymphoma/immunology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/immunology , Antigens, Neoplasm/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Cell Line, Tumor , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/genetics , Mice, SCIDABSTRACT
Innate immune cells in the colorectal cancer microenvironment mainly include macrophages, neutrophils, natural killer cells, dendritic cells and bone marrow-derived suppressor cells. They play a pivotal role in tumor initiation and progression through the secretion of diverse cytokines, chemokines, and other factors that govern these processes. Colorectal cancer is a common malignancy of the gastrointestinal tract, and understanding the role of innate immune cells in the microenvironment of CRC may help to improve therapeutic approaches to CRC and increase the good prognosis. In this review, we comprehensively explore the pivotal role of innate immune cells in the initiation and progression of colorectal cancer (CRC), alongside an extensive evaluation of the current landscape of innate immune cell-based immunotherapies, thereby offering valuable insights for future research strategies and clinical trials.
Subject(s)
Colorectal Neoplasms , Immunity, Innate , Tumor Microenvironment , Humans , Tumor Microenvironment/immunology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/therapy , Colorectal Neoplasms/pathology , Animals , Immunotherapy/methods , Killer Cells, Natural/immunologyABSTRACT
Breast cancer (BC) is the most commonly diagnosed cancer in women globally. Natural killer (NK) cells play a vital role in tumour immunosurveillance. This study aimed to establish a prognostic model using NK cell-related genes (NKRGs) by integrating single-cell transcriptomic data with machine learning. We identified 44 significantly expressed NKRGs involved in cytokine and T cell-related functions. Using 101 machine learning algorithms, the Lasso + RSF model showed the highest predictive accuracy with nine key NKRGs. We explored cell-to-cell communication using CellChat, assessed immune-related pathways and tumour microenvironment with gene set variation analysis and ssGSEA, and observed immune components by HE staining. Additionally, drug activity predictions identified potential therapies, and gene expression validation through immunohistochemistry and RNA-seq confirmed the clinical applicability of NKRGs. The nomogram showed high concordance between predicted and actual survival, linking higher tumour purity and risk scores to a reduced immune score. This NKRG-based model offers a novel approach for risk assessment and personalized treatment in BC, enhancing the potential of precision medicine.
Subject(s)
Breast Neoplasms , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Killer Cells, Natural , Machine Learning , Single-Cell Analysis , Transcriptome , Tumor Microenvironment , Humans , Breast Neoplasms/genetics , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Breast Neoplasms/diagnosis , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Female , Prognosis , Transcriptome/genetics , Single-Cell Analysis/methods , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Biomarkers, Tumor/genetics , NomogramsABSTRACT
Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19-20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , Antigens, CD20 , Complement Factor H , Leukemia, Lymphocytic, Chronic, B-Cell , Rituximab , Humans , Antigens, CD20/immunology , Antigens, CD20/genetics , Complement Factor H/immunology , Complement Factor H/metabolism , Complement Factor H/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Rituximab/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Cell Line, TumorABSTRACT
Activating antibody-dependent cellular cytotoxicity (ADCC) by targeting claudin-18 isoform 2 (CLDN18.2) using zolbetuximab, a monoclonal antibody against CLDN18.2, has been considered a promising novel therapeutic strategy for gastric cancer (GC). However, the impact of CLDN18.2 expression on natural killer (NK) cells and monocytes/macrophages-crucial effector cells of ADCC-in GC has not been fully investigated. In the present study, we assessed the impact of CLDN18.2 expression on clinical outcomes, molecular features, and the frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, in GC by analyzing our own GC cohorts. The expression of CLDN18.2 did not significantly impact clinical outcomes of GC patients, while it was significantly and positively associated with Epstein-Barr virus (EBV) status and PD-L1 expression. The frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, were comparable between CLDN18.2-positive and CLDN18.2-negative GCs. Importantly, both CLDN18.2 expression and the number of tumor-infiltrating NK cells were significantly higher in EBV-associated GC compared to other molecular subtypes. Our findings support the effectiveness of zolbetuximab in CLDN18.2-positive GC, and offer a novel insight into the treatment of this cancer type, highlighting its potential effectiveness for CLDN18.2-positive/EBV-associated GC.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , Claudins , Killer Cells, Natural , Stomach Neoplasms , Humans , Stomach Neoplasms/immunology , Stomach Neoplasms/pathology , Stomach Neoplasms/metabolism , Stomach Neoplasms/genetics , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Female , Claudins/metabolism , Claudins/genetics , Middle Aged , Aged , Macrophages/immunology , Macrophages/metabolism , Monocytes/immunology , Monocytes/metabolismABSTRACT
Multiple research studies have demonstrated the efficacy of lactic acid bacteria in boosting both innate and adaptive immune responses. We have created a Lactococcus lactis variant that produces a modified combination protein with Fms-like tyrosine kinase 3 ligand and co-stimulator O × 40 ligand, known as HuFOLactis. The genetically modified variant was purposely created to activate T cells, NK cells, and DC cells in a laboratory setting. Furthermore, we explored the possibility of using the tumor-penetrating peptide iRGD to deliver HuFOLactis-activated immune cells to hard-to-reach tumor areas. Following brief stimulation with HuFOLactis, immune cell phenotypes and functions were assessed using flow cytometry. Confocal microscopy was employed to demonstrate the infiltrative and cytotoxic capabilities of iRGD-modified HuFOLactis-activated immune cells within tumor spheroids. The efficacy of iRGD modified HuFOLactis-activated immune cells against tumors was assessed in xenograft mouse models. HuFOLactis treatment resulted in notable immune cell activation, demonstrated by elevated levels of CD25, CD69, and CD137. Additionally, these activated immune cells showed heightened cytokine production and enhanced cytotoxicity against MKN45 cell lines. Incorporation of the iRGD modification facilitated the infiltration of HuFOLactis-activated immune cells into multicellular spheroids (MCSs). Additionally, immune cells activated by HuFOLactis and modified with iRGD, in combination with anti-PD-1 treatment, effectively halted tumor growth and prolonged survival in a mouse model of gastric cancer.
Subject(s)
Lactococcus lactis , Animals , Mice , Lactococcus lactis/genetics , Oligopeptides/pharmacology , Humans , Cell Line, Tumor , Female , Killer Cells, Natural/immunology , T-Lymphocytes/immunology , Xenograft Model Antitumor Assays , Dendritic Cells/immunology , Dendritic Cells/drug effectsABSTRACT
INTRODUCTION: Glioblastoma (GBM) poses a significant challenge in oncology, with median survival times barely extending beyond a year due to resistance to standard therapies like temozolomide (TMZ). This study introduces a novel therapeutic strategy combining progesterone (Prog) and abiraterone (Abi) aimed at enhancing GBM treatment efficacy by modulating the tumor microenvironment and augmenting NK cell-mediated immunity. METHODS: We employed in vitro and in vivo GBM models to assess the effects of Prog and Abi on cell viability, proliferation, apoptosis, and the immune microenvironment. Techniques included cell viability assays, Glo-caspase 3/7 apoptosis assays, RNA-seq and qPCR for gene expression, Seahorse analysis for mitochondrial function, HPLC-MS for metabolomics analysis, and immune analysis by flow cytometry to quantify NK cell infiltration. RESULTS: Prog significantly reduced the IC50 of Abi in TMZ-resistant GBM cell, suggesting the enhanced cytotoxicity. Treatment induced greater apoptosis than either agent alone, suppressed tumor growth, and prolonged survival in mouse models. Notably, there was an increase in CD3-/CD19-/CD56+/NK1.1+ NK cell infiltration in treated tumors, indicating a shift towards an anti-tumor immune microenvironment. The combination therapy also resulted in a reduction of MGMT expression and a suppression of mitochondrial respiration and glycolysis in GBM cells. CONCLUSION: The combination of Prog and Abi represents a promising therapeutic approach for GBM, showing potential in suppressing tumor growth, extending survival, and modulating the immune microenvironment. These findings warrant further exploration into the clinical applicability of this strategy to improve outcomes for GBM patients.
Subject(s)
Glioblastoma , Killer Cells, Natural , Progesterone , Glioblastoma/drug therapy , Glioblastoma/pathology , Glioblastoma/metabolism , Glioblastoma/immunology , Humans , Mice , Animals , Killer Cells, Natural/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Progesterone/pharmacology , Androstenes/pharmacology , Androstenes/therapeutic use , Cell Line, Tumor , Xenograft Model Antitumor Assays , Apoptosis/drug effects , Tumor Microenvironment/drug effects , Brain Neoplasms/drug therapy , Brain Neoplasms/immunology , Brain Neoplasms/pathology , Brain Neoplasms/metabolism , Disease Models, AnimalABSTRACT
Asthma poses a major public health burden. While existing asthma drugs manage symptoms for many, some patients remain resistant. The lack of a cure, especially for severe asthma, compels exploration of novel therapies. Cancer immunotherapy successes with CAR-T cells suggest its potential for asthma treatment. Researchers are exploring various approaches for allergic diseases including membrane-bound IgE, IL-5, PD-L2, and CTLA-4 for asthma, and Dectin-1 for fungal asthma. NK cells offer several advantages over T cells for CAR-based immunotherapy. They offer key benefits: (1) HLA compatibility, meaning they can be used in a wider range of patients without the need for matching tissue types. (2) Minimal side effects (CRS and GVHD) due to their limited persistence and cytokine profile. (3) Scalability for "off-the-shelf" production from various sources. Several strategies have been introduced that highlight the superiority and challenges of CAR-NK cell therapy for asthma treatment including IL-10, IFN-γ, ADCC, perforin-granzyme, FASL, KIR, NCRs (NKP46), DAP, DNAM-1, TGF-ß, TNF-α, CCL, NKG2A, TF, and EGFR. Furthermore, we advocate for incorporating AI for CAR design optimization and CRISPR-Cas9 gene editing technology for precise gene manipulation to generate highly effective CAR constructs. This review will delve into the evolution and production of CAR designs, explore pre-clinical and clinical studies of CAR-based therapies in asthma, analyze strategies to optimize CAR-NK cell function, conduct a comparative analysis of CAR-T and CAR-NK cell therapy with their respective challenges, and finally present established novel CAR designs with promising potential for asthma treatment.
Subject(s)
Asthma , Killer Cells, Natural , Receptors, Chimeric Antigen , Humans , Asthma/therapy , Asthma/immunology , Killer Cells, Natural/immunology , Immunotherapy, Adoptive/methods , Immunotherapy/methods , AnimalsABSTRACT
BACKGROUND: Cancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity. METHODS: CSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry. RESULTS: We observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity. CONCLUSIONS: This study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.
Subject(s)
Cytotoxicity, Immunologic , Down-Regulation , Esophageal Neoplasms , Killer Cells, Natural , Neoplastic Stem Cells , Humans , Killer Cells, Natural/immunology , Esophageal Neoplasms/pathology , Esophageal Neoplasms/immunology , Esophageal Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Down-Regulation/drug effects , Cell Line, Tumor , Cytotoxicity, Immunologic/drug effects , GPI-Linked Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks, thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the 'leaky' DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic double-stranded DNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8+ T cell infiltration, activates macrophages and natural killer cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer and pancreatic ductal adenocarcinoma, which are refractory to ICBs, reveals that lower 53BP1 levels correlate with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.
Subject(s)
Membrane Proteins , Nucleotidyltransferases , Ovarian Neoplasms , Pancreatic Neoplasms , Tumor Suppressor p53-Binding Protein 1 , Tumor Suppressor p53-Binding Protein 1/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Female , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , Humans , Animals , Ovarian Neoplasms/immunology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Mice , Cell Line, Tumor , DNA Breaks, Double-Stranded , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , Signal Transduction , CD8-Positive T-Lymphocytes/immunology , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Mice, Inbred C57BL , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Mice, Knockout , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Immunity, InnateABSTRACT
Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.
Subject(s)
Antigens, CD , CD8-Positive T-Lymphocytes , Histocompatibility Antigens Class I , Lymphocyte Activation Gene 3 Protein , NK Cell Lectin-Like Receptor Subfamily D , Programmed Cell Death 1 Receptor , Animals , Antigens, CD/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Mice , Programmed Cell Death 1 Receptor/metabolism , NK Cell Lectin-Like Receptor Subfamily D/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , NK Cell Lectin-Like Receptor Subfamily C/metabolism , Mice, Inbred C57BL , High Mobility Group Proteins/metabolism , High Mobility Group Proteins/genetics , Cytotoxicity, Immunologic , Cell Proliferation , Killer Cells, Natural/metabolism , Killer Cells, Natural/immunologyABSTRACT
Introduction: The immunological characteristics that could protect children with coronavirus disease 2019 (COVID-19) from severe or fatal illnesses have not been fully understood yet. Methods: Here, we performed single-cell RNA sequencing (scRNA-seq) analysis on peripheral blood samples of 15 children (8 with COVID-19) and compared them to 18 adults (13 with COVID-19). Results: The child-adult integrated single cell data indicated that children with the disease presented a restrained response to type I interferon in most of the major immune cell types, along with suppression of upstream interferon regulatory factor and toll-like receptor expression in monocytes, which was confirmed by in vitro interferon stimulation assays. Unlike adult patients, children with COVID-19 showed lower frequencies of activated proinflammatory CD14+ monocytes, possibly explaining the rareness of cytokine storm in them. Notably, natural killer (NK) cells in pediatric patients displayed potent cytotoxicity with a rich expression of cytotoxic molecules and upregulated cytotoxic pathways, whereas the cellular senescence, along with the Notch signaling pathway, was significantly downregulated in NK cells, all suggesting more robust cytotoxicity in NK cells of children than adult patients that was further confirmed by CD107a degranulation assays. Lastly, a modest adaptive immune response was evident with more naïve T cells but less activated and proliferated T cells while less naïve B cells but more activated B cells in children over adult patients. Conclusion: Conclusively, this preliminary study revealed distinct cell frequency and activation status of major immune cell types, particularly more robust NK cell cytotoxicity in PBMC that might help protect children from severe COVID-19.
Subject(s)
COVID-19 , Killer Cells, Natural , SARS-CoV-2 , Single-Cell Analysis , Humans , COVID-19/immunology , Child , Adult , SARS-CoV-2/immunology , Male , Female , Killer Cells, Natural/immunology , Child, Preschool , Adolescent , Monocytes/immunology , Monocytes/metabolism , Middle Aged , Adaptive Immunity , Cytotoxicity, Immunologic , Young Adult , Interferon Type I/immunology , Cellular Senescence/immunologyABSTRACT
Production of large amounts of functional NK and CAR-NK cells represents one of the bottlenecks for NK-based immunotherapy. In this study, we developed a large-scale, reliable, and practicable NK and CAR-NK production using G-Rex 100M bioreactors, which depend on a gas-permeable membrane technology. This system holds large volumes of medium with enhanced oxygen delivery, creating conditions conducive to large-scale PBNK and CAR-NK expansions for cancer therapy. Both peripheral blood NK cells (PBNKs) and CAR-NKs expanded in these bioreactors retained similar immunophenotypes and exhibited comparable cytotoxicity towards hepatocellular carcinoma (HCC) cells akin to that of NK and CAR-NK cells expanded in G-Rex 6 well bioreactors. Importantly, cryopreservation minimally affected the cytotoxicity of NK cells expanded using the G-Rex 100M bioreactors, establishing a robust platform for scaled-up NK and CAR-NK cell production. This method is promising for the development of "off-the-shelf" NK cells, supporting the future clinical implementation of NK cell immunotherapy.
Subject(s)
Bioreactors , Immunotherapy, Adoptive , Killer Cells, Natural , Receptors, Chimeric Antigen , Killer Cells, Natural/immunology , Humans , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Immunotherapy, Adoptive/methods , Cell Culture Techniques/methods , Cytotoxicity, Immunologic , Cell Line, Tumor , Cell Proliferation , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/therapy , Liver Neoplasms/immunology , Liver Neoplasms/therapyABSTRACT
Hemophagocytic lymphohistiocytosis (HLH) is a severe cytokine storm syndrome (CSS), which until the turn of the century, was barely known but is now receiving increased attention. The history of HLH dates back to 1939 when it was first described in adults, to be followed in 1952 by the first description of its primary, familial form in children. Secondary forms of HLH are far more frequent and occur with infections, malignancies, metabolic diseases, iatrogenic immune suppression, and autoinflammatory/autoimmune diseases. Identification of the genetic defects leading to the defective function of natural killer (NK) cells and cytotoxic T cells as well as the corresponding mouse models have revolutionized our understanding of HLH and of immune function. Diagnosis relies on clinical and laboratory criteria; functional and genetic tests can help separate primary from secondary forms. Treatment with immunochemotherapy and hematopoietic stem cell transplantation has considerably improved survival in children with primary HLH, a formerly uniformly fatal disease.
Subject(s)
Lymphohistiocytosis, Hemophagocytic , Lymphohistiocytosis, Hemophagocytic/therapy , Lymphohistiocytosis, Hemophagocytic/diagnosis , Lymphohistiocytosis, Hemophagocytic/genetics , Lymphohistiocytosis, Hemophagocytic/immunology , Humans , History, 20th Century , Animals , History, 21st Century , Killer Cells, Natural/immunology , Hematopoietic Stem Cell TransplantationABSTRACT
Macrophage activation syndrome (MAS) is a life-threatening episode of hyperinflammation driven by excessive activation and expansion of T cells (mainly CD8) and hemophagocytic macrophages producing proinflammatory cytokines. MAS has been reported in association with almost every rheumatic disease, but it is by far most common in systemic juvenile idiopathic arthritis (SJIA). Clinically, MAS is similar to familial or primary hemophagocytic lymphohistiocytosis (pHLH), a group of rare autosomal recessive disorders linked to various genetic defects all affecting the perforin-mediated cytolytic pathway employed by NK cells and cytotoxic CD8 T lymphocytes. Decreased cytolytic activity in pHLH patients leads to prolonged survival of target cells associated with increased production of proinflammatory cytokines that overstimulate macrophages. The resulting cytokine storm is believed to be responsible for the frequently fatal multiorgan system failure seen in MAS. Whole exome sequencing as well as targeted sequencing of pHLH-associated genes in patients with SJIA-associated MAS demonstrated increased "burden" of rare protein-altering variants affecting the cytolytic pathway compared to healthy controls, suggesting that as in pHLH, genetic variability in the cytolytic pathway contributes to MAS predisposition. Functional studies of some of the novel variants have shown that even in a heterozygous state, their presence partially reduces cytolytic activity that may lead to increased cytokine production.