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1
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Meng XM, Wang L, Nikolic-Paterson DJ, Lan HY. Innate immune cells in acute and chronic kidney disease. Nat Rev Nephrol 2025:10.1038/s41581-025-00958-x. [PMID: 40263532 DOI: 10.1038/s41581-025-00958-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/24/2025] [Indexed: 04/24/2025]
Abstract
Acute kidney injury (AKI) and chronic kidney disease (CKD) are inter-related clinical and pathophysiological disorders. Cells of the innate immune system, such as granulocytes and macrophages, can induce AKI through the secretion of pro-inflammatory mediators such as cytokines, chemokines and enzymes, and the release of extracellular traps. In addition, macrophages and dendritic cells can drive the progression of CKD through a wide range of pro-inflammatory and pro-fibrotic mechanisms, and by regulation of the adaptive immune response. However, innate immune cells can also promote kidney repair after acute injury. These actions highlight the multifaceted nature of the way by which innate immune cells respond to signals within the kidney microenvironment, including interaction with the complement and coagulation cascades, cells of the adaptive immune system, intrinsic renal cells and infiltrating mesenchymal cells. The factors and mechanisms that underpin the ability of innate immune cells to contribute to renal injury or repair and to drive the progression of CKD are of great interest for understanding disease processes and for developing new therapeutic approaches to limit AKI and the AKI-to-CKD transition.
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Affiliation(s)
- Xiao-Ming Meng
- Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, the Key Laboratory of Anti-inflammatory of Immune Medicines, Ministry of Education, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, China
| | - Li Wang
- Research Center of Integrated Traditional Chinese and Western Medicine, the Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, China
| | - David J Nikolic-Paterson
- Department of Nephrology, Monash Medical Centre and Monash University Centre for Inflammatory Diseases, Melbourne, Victoria, Australia
| | - Hui-Yao Lan
- Research Center of Integrated Traditional Chinese and Western Medicine, the Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, China.
- Departments of Medicine & Therapeutics, the Chinese University of Hong Kong, Hong Kong, and Guangdong-Hong Kong Joint Laboratory for Immunological and Genetic Kidney Disease, Guangdong Academy of Medical Science, Guangdong Provincial People's Hospital, Guangzhou, China.
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2
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Vincken R, Armendáriz-Martínez U, Ruiz-Sáenz A. ADCC: the rock band led by therapeutic antibodies, tumor and immune cells. Front Immunol 2025; 16:1548292. [PMID: 40308580 PMCID: PMC12040827 DOI: 10.3389/fimmu.2025.1548292] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Accepted: 03/27/2025] [Indexed: 05/02/2025] Open
Abstract
Antibody-dependent cellular cytotoxicity (ADCC) is a critical mechanism by which therapeutic antibodies leverage the immune system to target and eliminate cancer cells. The key agents of ADCC are natural killer (NK) cells, specifically targeting antibody-covered cancer cells through the CD16 receptor. While other immune cells and Fc receptors can contribute and enhance ADCC, NK cells and the CD16 receptor are crucial for the efficacy of cancer therapies such as trastuzumab, cetuximab and rituximab. Co-culture assays are essential for understanding the mechanisms of these therapies, overcoming resistance and optimizing novel therapeutic antibodies. This review highlights the importance of measuring ADCC to assess the efficacy of therapeutic antibodies. Here we also present the various in vitro models and assay methodologies available for studying ADCC, comparing the strengths and limitations of approaches like using PBMCs to better reflect real-life conditions or NK cell lines for standardization. It also covers different readouts for ADCC, either focusing on effector cells activation, including reporter and degranulation assays or in the target cell killing, including different molecule release assays, flow cytometry and immunofluorescence techniques. Selecting the best model for studying ADCC is crucial for the translational significance of therapeutic antibody research.
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Affiliation(s)
- Roos Vincken
- Department of Cell Biology, Erasmus University Medical Center Rotterdam, CN, Rotterdam, Netherlands
- Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
| | - Uxue Armendáriz-Martínez
- Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
| | - Ana Ruiz-Sáenz
- Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
- IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
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3
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Felgueres MJ, Esteso G, García-Jiménez ÁF, Benguría A, Vázquez E, Aguiló N, Puentes E, Dopazo A, Murillo I, Martín C, Rodríguez E, Reyburn HT, Valés-Gómez M. Cytolytic γδ T-cells and IFNγ-producing CD4-lymphocytes characterise the early response to MTBVAC tuberculosis vaccine. NPJ Vaccines 2025; 10:58. [PMID: 40155627 PMCID: PMC11953372 DOI: 10.1038/s41541-025-01110-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2024] [Accepted: 03/14/2025] [Indexed: 04/01/2025] Open
Abstract
Infection with Mycobacterium tuberculosis (Mtb) can produce a wide spectrum of clinical manifestations, ranging from active tuberculosis (TB) to asymptomatic latent infection. Although CD4 T-cells are key immune effectors to control TB, early after infection, the innate immune response must play a role in tackling the disease. Here, we performed in-depth analyses of the acute immune response to MTBVAC, a candidate vaccine engineered from Mtb with the aim of protecting adults from pulmonary TB disease, still a major global challenge. scRNA-seq shows expansion of CD4+ and cytotoxic γδ T-cells, data confirmed by flow cytometry. CD4 T-cells exhibited lower HLA-DR and higher L-selectin expression, compared to BCG-stimulation, indicating differential activation or dynamics. Importantly, MTBVAC-activated γδ T-cells had a unique cytotoxic CD16+GZMB+ phenotype, reminiscent of effector cells found in Mtb positive individuals controlling infection. IFN-γ and TNF-α were released in cultures, while IL-17A/F were almost undetectable.
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Affiliation(s)
- María-José Felgueres
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain
| | - Gloria Esteso
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain
| | - Álvaro F García-Jiménez
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain
| | - Alberto Benguría
- Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Enrique Vázquez
- Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Nacho Aguiló
- Department of Microbiology, Pediatrics, Radiology and Public Health of the University of Zaragoza and Centro Investigación en Red de Enfermedades Respiratorias CIBERES, ISCIII, Zaragoza, Spain
| | - Eugenia Puentes
- Clinical Research and Research & Development Departments, Biofabri, Zendal Group, O'Porriño, Pontevedra, Spain
| | - Ana Dopazo
- Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- CIBER de Enfermedades Cardiovasculares, (CIBERCV), Madrid, Spain
| | - Ingrid Murillo
- Clinical Research and Research & Development Departments, Biofabri, Zendal Group, O'Porriño, Pontevedra, Spain
| | - Carlos Martín
- Department of Microbiology, Pediatrics, Radiology and Public Health of the University of Zaragoza and Centro Investigación en Red de Enfermedades Respiratorias CIBERES, ISCIII, Zaragoza, Spain
| | - Esteban Rodríguez
- Clinical Research and Research & Development Departments, Biofabri, Zendal Group, O'Porriño, Pontevedra, Spain
| | - Hugh T Reyburn
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain
| | - Mar Valés-Gómez
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain.
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4
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Zhang N, Liang Y, Meng YQ, Li YC, Lu X, Li L, Ye T. Analysis and identification of potential biomarkers for dysfunctional uterine bleeding. J Reprod Immunol 2025; 168:104427. [PMID: 39862473 DOI: 10.1016/j.jri.2025.104427] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 12/04/2024] [Accepted: 01/05/2025] [Indexed: 01/27/2025]
Abstract
Clinical evidence increasingly suggests that traditional treatments for dysfunctional uterine bleeding (DUB) have limited success. In this study, blood samples from 10 DUB patients and 10 healthy controls were collected for transcriptome sequencing. Then, the differentially expressed genes (DEGs) were screened and crossed with the DUB-related module genes to obtain the target genes. These target genes were analyzed for functional enrichment. Further, the biomarkers were screened by protein-protein interaction (PPI) analysis and analyzed by the gene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA). To explore the pathogenesis of DUB, immune microenvironment analyses were also performed. Potential drugs targeting these biomarkers were predicted for clinical treatment. The expression of these biomarkers was validated using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that, a total of 754 target genes were found to be related to cell proliferation and senescence. Five biomarkers-CENPE, KIF11, PIK3R1, SMC3, and SMC4-were identified, all of which were down-regulated in the DUB group, and most of these findings were confirmed by qRT-PCR. Notably, CENPE expression showed a negative association with activated NK cells and a positive association with resting NK cells. In addition, 44 potential drugs were predicted for DUB treatment. In conclusion, five DUB biomarkers were identified, enhancing understanding of gene regulation in DUB and providing a theoretical foundation for clinical diagnosis and treatment.
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Affiliation(s)
- N Zhang
- Department of Pharmacy, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 50001, China
| | - Y Liang
- Department of Gynaecology, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China
| | - Y Q Meng
- Department of Gynaecology, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China
| | - Y C Li
- Department of Pharmacy, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 50001, China
| | - X Lu
- College of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China
| | - L Li
- Department of Pharmacy, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 50001, China.
| | - T Ye
- Department of Chinese Medicine Rehabilitation, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 50001, China.
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5
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Park S, Paek JH, Colville MJ, Huang LT, Struzyk AP, Womack SJ, Neelamegham S, Reesink HL, Paszek MJ. Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.05.627089. [PMID: 39677754 PMCID: PMC11643053 DOI: 10.1101/2024.12.05.627089] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
The abnormally thick glycocalyx of cancer cells can provide a physical barrier to immune cell recognition and effective immunotherapy. Here, we demonstrate an optical method based on Scanning Angle Interference Microscopy (SAIM) for the screening of therapeutic agents that can disrupt the glycocalyx layer as a strategy to improve anti-cancer immune responses. We developed a new membrane labeling strategy utilizing leucine zipper pairs to fluorescently mark the glycocalyx layer boundary for precise and robust measurement of glycocalyx thickness with SAIM. Using this platform, we evaluated the effects of glycosylation inhibitors and targeted enzymatic degraders of the glycocalyx, with particular focus on strategies for cholangiocarcinoma (CCA), a highly lethal malignancy with limited therapeutic options. We found that CCA had the highest mean expression of the cancer-associated mucin, MUC1, across all cancers represented in the cancer cell line encyclopedia. Pharmacological inhibitors of mucin-type O-glycosylation and mucin-specific proteases, such as StcE, could dramatically reduce the glycocalyx layer in the YSCCC model of intrahepatic CCA. Motivated by these findings, we engineered Natural Killer (NK) cells tethered with StcE to enhance NK cell-mediated cytotoxicity against CCA. In a CCA xenograft model, these engineered NK cells demonstrated superior anti-tumor efficacy compared to wild-type NK cells, with no observable adverse effects. Our findings not only provide a reliable imaging-based screening platform for evaluating glycocalyx-targeting pharmacological interventions but also offer mechanistic insights into how CCA may avoid immune elimination through fortification of the glycocalyx layer with mucins. Additionally, this work presents a novel therapeutic strategy for mucin-overexpressing cancers, potentially improving immunotherapy efficacy across various cancer types.
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Affiliation(s)
- Sangwoo Park
- Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
- Current address: Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- These authors contributed equally to this work
| | - Justin H. Paek
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
- These authors contributed equally to this work
| | - Marshall J. Colville
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | - Ling-Ting Huang
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | - Audrey P. Struzyk
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Sydney J. Womack
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | | | - Heidi L. Reesink
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Matthew J. Paszek
- Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
- Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, USA
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Park S, Choi S, Shimpi AA, Estroff LA, Fischbach C, Paszek MJ. Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2024; 36:e2311505. [PMID: 38279892 PMCID: PMC11471288 DOI: 10.1002/adma.202311505] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Revised: 01/14/2024] [Indexed: 01/29/2024]
Abstract
Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.
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Affiliation(s)
- Sangwoo Park
- Graduate Field of Biophysics, Cornell University, Ithaca, NY 14853, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA
| | - Siyoung Choi
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA
| | - Adrian A. Shimpi
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA
| | - Lara A. Estroff
- Department of Materials Science and Engineering, Cornell University, Ithaca, NY, 14853, USA
- Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, 14853, USA
| | - Claudia Fischbach
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA
- Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, 14853, USA
| | - Matthew J. Paszek
- Graduate Field of Biophysics, Cornell University, Ithaca, NY 14853, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA
- Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, 14853, USA
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7
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Felgueres MJ, Esteso G, Aguiló N, Valés-Gómez M. Selective expansion of anti-tumor innate lymphocytes in long-term cultures after a single BCG pulse. Methods Cell Biol 2024; 190:203-221. [PMID: 39515880 DOI: 10.1016/bs.mcb.2024.07.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2024]
Abstract
Natural Killer (NK) cells are cytotoxic lymphocytes involved in the recognition of pathogen-infected and cancer cells. NK cells are very attractive as cell therapy tools because they are neither restricted by donor compatibility nor do they cause toxicity. Although their anti-tumor role has been long known, for development of NK-based therapies it is important to select the appropriate subpopulation. Similarly, non-MHC restricted T cells, in particular γδ T cells, have also been proposed as novel weapons against cancer. Here, we describe a new approach for production and characterization of anti-tumor innate lymphocyte cultures, containing mainly NK and γδ T cells, based on stimulation of peripheral blood mononuclear cells (PBMC) with BCG (Bacillus Calmette-Guérin), the tuberculosis vaccine, which is also successfully used to treat non-muscle invasive bladder cancer. Anti-tumor innate lymphocytes specifically proliferate from BCG-primed PBMC and can be cultured for weeks in low doses of IL12, IL15 and IL21. These cells kill a wide range of tumors and remain functional for weeks, with minimal manipulation. The phenotypic analysis of these cultures by multi-parametric flow cytometry is explained. Functional assays, including lymphocyte degranulation, cytokine production and radioactive isotope-free specific lysis experiments are also described.
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Affiliation(s)
- María-José Felgueres
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain
| | - Gloria Esteso
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain
| | - Nacho Aguiló
- Department of Microbiology, Pediatrics, Radiology and Public Health of the University of Zaragoza, IIS Aragon, CIBER de Enfermedades Respiratorias, Zaragoza, Spain
| | - Mar Valés-Gómez
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Madrid, Spain.
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8
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Wang Q, Nag D, Baldwin SL, Coler RN, McNamara RP. Antibodies as key mediators of protection against Mycobacterium tuberculosis. Front Immunol 2024; 15:1430955. [PMID: 39286260 PMCID: PMC11402706 DOI: 10.3389/fimmu.2024.1430955] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Accepted: 08/13/2024] [Indexed: 09/19/2024] Open
Abstract
Tuberculosis (TB) is caused by infection with the bacterial pathogen Mycobacterium tuberculosis (M.tb) in the respiratory tract. There was an estimated 10.6 million people newly diagnosed with TB, and there were approximately 1.3 million deaths caused by TB in 2022. Although the global prevalence of TB has remained high for decades and is an annual leading cause of death attributed to infectious diseases, only one vaccine, Bacillus Calmette-Guérin (BCG), has been approved so far to prevent/attenuate TB disease. Correlates of protection or immunological mechanisms that are needed to control M.tb remain unknown. The protective role of antibodies after BCG vaccination has also remained largely unclear; however, recent studies have provided evidence for their involvement in protection against disease, as biomarkers for the state of infection, and as potential predictors of outcomes. Interestingly, the antibodies generated post-vaccination with BCG are linked to the activation of innate immune cascades, providing further evidence that antibody effector functions are critical for protection against respiratory pathogens such as M.tb. In this review, we aim to provide current knowledge of antibody application in TB diagnosis, prevention, and treatment. Particularly, this review will focus on 1) The role of antibodies in preventing M.tb infections through preventing Mtb adherence to epithelium, antibody-mediated phagocytosis, and antibody-mediated cellular cytotoxicity; 2) The M.tb-directed antibody response generated after vaccination and how humoral profiles with different glycosylation patterns of these antibodies are linked with protection against the disease state; and 3) How antibody-mediated immunity against M.tb can be further explored as early diagnosis biomarkers and different detection methods to combat the global M.tb burden. Broadening the paradigm of differentiated antibody profiling and antibody-based detection during TB disease progression offers new directions for diagnosis, treatment, and preventative strategies. This approach involves linking the aforementioned humoral responses with the disease state, progression, and clearance.
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Affiliation(s)
- Qixin Wang
- Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA, United States
| | - Deepika Nag
- Seattle Children’s Research Institute, Center for Global Infectious Disease Research, Seattle, WA, United States
| | - Susan L. Baldwin
- Seattle Children’s Research Institute, Center for Global Infectious Disease Research, Seattle, WA, United States
| | - Rhea N. Coler
- Seattle Children’s Research Institute, Center for Global Infectious Disease Research, Seattle, WA, United States
- Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, United States
- Department of Global Health, University of Washington, Seattle, WA, United States
| | - Ryan P. McNamara
- Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA, United States
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Manohar SM. Shedding Light on Intracellular Proteins using Flow Cytometry. Cell Biochem Biophys 2024; 82:1693-1707. [PMID: 38831173 DOI: 10.1007/s12013-024-01338-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/24/2024] [Indexed: 06/05/2024]
Abstract
Intracellular protein abundance is routinely measured in mammalian cells using population-based techniques such as western blotting which fail to capture single cell protein levels or using fluorescence microscopy which is although suitable for single cell protein detection but not for rapid analysis of large no. of cells. Flow cytometry offers rapid, high-throughput, multiparameter-based analysis of intracellular protein expression in statistically significant no. of cells at single cell resolution. In past few decades, customized assays have been developed for flow cytometric detection of specific intracellular proteins. This review discusses the scope of flow cytometry for intracellular protein detection in mammalian cells along with specific applications. Technological advancements to overcome the limitations of traditional flow cytometry for the same are also discussed.
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Affiliation(s)
- Sonal M Manohar
- Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS (Deemed-to-be) University, Vile Parle (West), Mumbai, 400056, India.
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10
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Felgueres MJ, Esteso G, García-Jiménez ÁF, Dopazo A, Aguiló N, Mestre-Durán C, Martínez-Piñeiro L, Pérez-Martínez A, Reyburn HT, Valés-Gómez M. BCG priming followed by a novel interleukin combination activates Natural Killer cells to selectively proliferate and become anti-tumour long-lived effectors. Sci Rep 2024; 14:13133. [PMID: 38849432 PMCID: PMC11161620 DOI: 10.1038/s41598-024-62968-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Accepted: 05/23/2024] [Indexed: 06/09/2024] Open
Abstract
The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients' bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.
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Affiliation(s)
- María-José Felgueres
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Darwin, 3, 28049, Madrid, Spain
| | - Gloria Esteso
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Darwin, 3, 28049, Madrid, Spain
| | - Álvaro F García-Jiménez
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Darwin, 3, 28049, Madrid, Spain
| | - Ana Dopazo
- Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Nacho Aguiló
- Department of Microbiology, Pediatrics, Radiology and Public Health of the University of Zaragoza, IIS Aragon, CIBER de Enfermedades Respiratorias, Zaragoza, Spain
| | - Carmen Mestre-Durán
- Translational Research in Pediatric Oncology, Hematopoietic Transplantation and Cell Therapy, IdiPAZ, and Pediatric Hemato-Oncology, Hospital Universitario La Paz, Madrid, Spain
- IdiPAZ-CNIO Pediatric Onco-Hematology Clinical Research Unit, Spanish National Cancer Research Centre (CNIO), 28049, Madrid, Spain
| | - Luis Martínez-Piñeiro
- Urology Department and Hospital La Paz Institute for Health Research (IdiPAZ), La Paz University Hospital, Madrid, Spain
| | - Antonio Pérez-Martínez
- Translational Research in Pediatric Oncology, Hematopoietic Transplantation and Cell Therapy, IdiPAZ, and Pediatric Hemato-Oncology, Hospital Universitario La Paz, Madrid, Spain
- IdiPAZ-CNIO Pediatric Onco-Hematology Clinical Research Unit, Spanish National Cancer Research Centre (CNIO), 28049, Madrid, Spain
- Pediatric Department, Autonomous University of Madrid, Madrid, Spain
| | - Hugh T Reyburn
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Darwin, 3, 28049, Madrid, Spain
| | - Mar Valés-Gómez
- Department of Immunology and Oncology, National Centre for Biotechnology, Spanish National Research Council (CNB-CSIC), Darwin, 3, 28049, Madrid, Spain.
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11
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Park S, Colville MJ, Paek JH, Shurer CR, Singh A, Secor EJ, Sailer CJ, Huang LT, Kuo JCH, Goudge MC, Su J, Kim M, DeLisa MP, Neelamegham S, Lammerding J, Zipfel WR, Fischbach C, Reesink HL, Paszek MJ. Immunoengineering can overcome the glycocalyx armour of cancer cells. NATURE MATERIALS 2024; 23:429-438. [PMID: 38361041 PMCID: PMC11471287 DOI: 10.1038/s41563-024-01808-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Accepted: 01/03/2024] [Indexed: 02/17/2024]
Abstract
Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.
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Affiliation(s)
- Sangwoo Park
- Field of Biophysics, Cornell University, Ithaca, NY, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | - Marshall J Colville
- Field of Biophysics, Cornell University, Ithaca, NY, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | - Justin H Paek
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Carolyn R Shurer
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | - Arun Singh
- State University of New York, Buffalo, NY, USA
| | - Erica J Secor
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Cooper J Sailer
- Department of Pathology, University of Rochester Medical Center, Rochester, NY, USA
| | - Ling-Ting Huang
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | - Joe Chin-Hun Kuo
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | - Marc C Goudge
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Jin Su
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Minsoo Kim
- Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA
| | - Matthew P DeLisa
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
| | | | - Jan Lammerding
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
- Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA
| | - Warren R Zipfel
- Field of Biophysics, Cornell University, Ithaca, NY, USA
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Claudia Fischbach
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Heidi L Reesink
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
| | - Matthew J Paszek
- Field of Biophysics, Cornell University, Ithaca, NY, USA.
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
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12
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Karaselek MA, Kurar E, Keleş S, Güner ŞN, Reisli İ. Association of NK cell subsets and cytotoxicity with FCGR3A gene polymorphism in functional NK cell deficiency. REVISTA DA ASSOCIACAO MEDICA BRASILEIRA (1992) 2024; 70:e20230872. [PMID: 38422319 PMCID: PMC10903273 DOI: 10.1590/1806-9282.20230872] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/29/2023] [Accepted: 09/07/2023] [Indexed: 03/02/2024]
Abstract
OBJECTIVE The purpose of this study was to assess the association between clinical, laboratory, and functional analyses and polymorphism in the FCGR3A gene in individuals with functional NK cell deficiency. METHODS A total of 15 functional NK cell deficiency patients and 10 age-matched healthy controls underwent NK cell subgroup, cytotoxicity, and FCGR3A whole-exome analysis with next-generation sequencing. RESULTS Three different NK cell subsets (CD56brightCD16neg, CD56brightCD16int, and CD56dimCD16hi) were identified. No statistically significant difference was found in the ratio of CD56brightCD16neg cells between patients and controls. CD56brightCD16int and CD56dimCD16hi ratios were found to be significantly lower in patients. As a result of NK cell cytotoxicity analysis, a proportional decrease of K562 amount between patients and controls was found to be statistically significant (p<0.001). In the FCGR3A whole-exome analysis, all patients were found to be homozygous mutant for the c.526G > T (p.V176F) in exon 4, while three patients were homozygous wild type and 12 patients were heterozygous for the c.197T>A (p.L66H) in exon 3. CONCLUSION In this study, a group of pediatric patients with suspected functional NK cell deficiency were evaluated and the findings indicated that NK subsets, cytotoxicity results, and FCGR3A gene polymorphism were found to be correlated with the clinical features. We conclude that this kind of study might contribute to follow-up the patients in time.
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Affiliation(s)
- Mehmet Ali Karaselek
- Necmettin Erbakan University, Faculty of Medicine, Department of Medical Biology - Konya, Turkey
| | - Ercan Kurar
- Necmettin Erbakan University, Faculty of Medicine, Department of Medical Biology - Konya, Turkey
| | - Sevgi Keleş
- Necmettin Erbakan University, Faculty of Medicine, Department of Pediatric Immunology and Allergy - Konya, Turkey
| | - Şükrü Nail Güner
- Necmettin Erbakan University, Faculty of Medicine, Department of Pediatric Immunology and Allergy - Konya, Turkey
| | - İsmail Reisli
- Necmettin Erbakan University, Faculty of Medicine, Department of Pediatric Immunology and Allergy - Konya, Turkey
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Park S, Choi S, Shimpi AA, Estroff LA, Fischbach C, Paszek MJ. COLLAGEN MINERALIZATION DECREASES NK CELL-MEDIATED CYTOTOXICITY OF BREAST CANCER CELLS VIA INCREASED GLYCOCALYX THICKNESS. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.20.576377. [PMID: 38328161 PMCID: PMC10849468 DOI: 10.1101/2024.01.20.576377] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/09/2024]
Abstract
Skeletal metastasis is common in patients with advanced breast cancer, and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow, but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, we have utilized a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. Our results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increased their glycocalyx thickness while enhancing resistance to attack by Natural Killer (NK) cells. These changes were functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, our results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.
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Affiliation(s)
- Sangwoo Park
- Graduate Field of Biophysics, Cornell University, Ithaca, NY 14853, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA
| | - Siyoung Choi
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA
| | - Adrian A. Shimpi
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA
| | - Lara A. Estroff
- Department of Materials Science and Engineering, Cornell University, Ithaca, NY, 14853, USA
- Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, 14853, USA
| | - Claudia Fischbach
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA
- Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, 14853, USA
| | - Matthew J. Paszek
- Graduate Field of Biophysics, Cornell University, Ithaca, NY 14853, USA
- Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA
- Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, 14853, USA
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Rosado FG, Gopal P. Laboratory Features and Pathology of Cytokine Storm Syndromes. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 1448:43-58. [PMID: 39117807 DOI: 10.1007/978-3-031-59815-9_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/10/2024]
Abstract
The laboratory diagnosis of cytokine storm syndromes (CSSs), i.e., hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS), is often challenging. The laboratory features using routinely available tests lack specificity, whereas confirmatory testing is available in only few laboratories in the United States. The disease mechanisms are still largely unclear, particularly in adults. In this chapter, the pathogenesis of CSSs, their associated laboratory findings, and recommended diagnostic strategies are reviewed.
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Affiliation(s)
- Flavia G Rosado
- Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Purva Gopal
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA
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15
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Hanton AJ, Waddell LA, Hope JC, Gray M, Wu Z. Bovine NK subsets in the afferent lymph and lymph nodes have distinct expression of naïve and activation-associated cell surface expressed molecules, and are differentially stimulated by BCG vaccination. Vet Immunol Immunopathol 2023; 266:110682. [PMID: 38000215 DOI: 10.1016/j.vetimm.2023.110682] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2023] [Revised: 11/10/2023] [Accepted: 11/14/2023] [Indexed: 11/26/2023]
Abstract
Bovine natural killer (bNK) cells are heterogeneous cell populations defined by constitutive expression of the natural cytotoxicity receptor, NKp46 (CD335). Two major subsets of bNK cells, classified by differential expression of CD2, display divergent functions in innate immunity, and are hypothesised to contribute to adaptive immunity following vaccination. Here we characterised phenotypic variation of bNK cells within afferent lymph and lymph node (LN) tissues and between CD2+ and CD2- bNK subsets, and report phenotypic changes induced by BCG vaccination. CD2- bNK cells, which dominate in the afferent lymph and LN, displayed lower expression of the activation marker CD25 within the LN, with CD25+ cells being less than half as frequent as in afferent lymph. Furthermore, we found bNK cells had a lower expression of CD45RB, associated in cattle with naïve cell status, within LN compared to afferent lymph. Following BCG vaccination, bNK cells in afferent lymph draining the vaccination site showed increased CD2-CD25+ frequencies and increased expression of CD25 on CD2+ bNK cells, although the frequency of these cells remained unchanged. In summary, we provide an overview of the phenotype of bNK cells within bovine lymphatic tissues, and provide an indication of how subsets may diverge following BCG vaccination.
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Affiliation(s)
| | | | - Jayne C Hope
- The Roslin Institute, University of Edinburgh, EH25 9RG, UK
| | - Mark Gray
- Royal (Dick) School of Veterinary Studies, University of Edinburgh, EH25 9RG, UK
| | - Zhiguang Wu
- The Roslin Institute, University of Edinburgh, EH25 9RG, UK.
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16
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Dawodu D, Sand S, Nikolouli E, Werfel T, Mommert S. The mRNA expression and secretion of granzyme B are up-regulated via the histamine H2 receptor in human CD4 + T cells. Inflamm Res 2023; 72:1525-1538. [PMID: 37470818 PMCID: PMC10499701 DOI: 10.1007/s00011-023-01759-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 06/01/2023] [Accepted: 06/09/2023] [Indexed: 07/21/2023] Open
Abstract
INTRODUCTION Granzyme B (GZMB), a serine protease with cytotoxic and immunomodulatory functions, shows elevated levels in blood plasma of patients with atopic dermatitis (AD). It has been observed that GZMB expression in CD4+ and CD8+ T cells is higher in lesional skin in AD than in healthy skin. Since histamine is present in high concentration in the skin of AD patients, we investigated the regulation of GZMB in human CD4+ T cells by histamine. METHODS Naïve CD4+ T cells polarized into Th2 cells, total CD4+ T cells treated with IL-4 for 72 h and CD4+ T cells isolated from healthy donors and AD patients were investigated. The cells were stimulated with histamine or with different histamine-receptor agonists. Gene expression was evaluated by RNA-Seq. GZMB mRNA expression was detected by quantitative real time PCR, whereas GZMB secretion was measured by ELISpot and ELISA. T cell degranulation was evaluated by flow cytometry using CD107a surface expression as a degranulation marker. RESULTS By RNA-Seq, we identified the up-regulation of various genes of the cytotoxic pathway, in particular of GZMB, by histamine in Th2-polarized CD4+ T cells. In Th2-polarized CD4+ T cells and in CD4+ T cells activated by IL-4 the mRNA expression of GZMB was significantly up-regulated by histamine and by histamine H2 receptor (H2R) agonists. The induction of GZMB secretion by histamine was significantly higher in CD4+ T cells from AD patients than in those from healthy donors. CD107a surface expression was up-regulated by trend in response to histamine in Th2-polarized CD4+ T cells. CONCLUSION Our findings may help to elucidate novel mechanisms of the H2R and to achieve a better understanding of the role of GZMB in the pathogenesis of AD.
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Affiliation(s)
- Damilola Dawodu
- Department of Dermatology and Allergy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Sophie Sand
- Department of Dermatology and Allergy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Eirini Nikolouli
- Department of Dermatology and Allergy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Thomas Werfel
- Department of Dermatology and Allergy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany
| | - Susanne Mommert
- Department of Dermatology and Allergy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
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17
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Carannante V, Wiklund M, Önfelt B. In vitro models to study natural killer cell dynamics in the tumor microenvironment. Front Immunol 2023; 14:1135148. [PMID: 37457703 PMCID: PMC10338882 DOI: 10.3389/fimmu.2023.1135148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Accepted: 06/05/2023] [Indexed: 07/18/2023] Open
Abstract
Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field.
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Affiliation(s)
- Valentina Carannante
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden
| | - Martin Wiklund
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden
| | - Björn Önfelt
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden
- Center for Infectious Medicine, Department of Medicine Huddinge, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden
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18
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Beelen NA, Ehlers FAI, Kooreman LFS, Bos GMJ, Wieten L. An in vitro model to monitor natural killer cell effector functions against breast cancer cells derived from human tumor tissue. Methods Cell Biol 2023; 173:133-153. [PMID: 36653080 DOI: 10.1016/bs.mcb.2022.05.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Adoptive natural killer (NK) cell-based immunotherapy poses a promising treatment approach in cancer. Despite minimal toxicities associated with NK cell infusion, the potential of NK cell therapy is inhibited by the immunosuppressive tumor microenvironment (TME). Multiple approaches to improve anti-cancer NK cell effector functions are being investigated. While much of this preclinical research is currently performed with commercially available tumor cell lines, this approach lacks the influence of the TME and heterogeneity of the primary tumor in patients. Here, we describe a comprehensive protocol for NK cell cytotoxicity- and degranulation assays against tumor cells derived from primary breast cancer tissue. Treatments to boost NK cell anti-tumor effector functions can be implemented in this model. Moreover, by using culture supernatants in follow up assays or by including additional cell types in the co-culture system, other NK cell effector mechanisms that further orchestrate innate and adaptive immunity could be studied.
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Affiliation(s)
- Nicky A Beelen
- Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center, Maastricht, The Netherlands; GROW-School for Oncology and Reproduction, Maastricht University, Maastricht, The Netherlands; Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands
| | - Femke A I Ehlers
- Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center, Maastricht, The Netherlands; GROW-School for Oncology and Reproduction, Maastricht University, Maastricht, The Netherlands; Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands
| | - Loes F S Kooreman
- GROW-School for Oncology and Reproduction, Maastricht University, Maastricht, The Netherlands; Department of Pathology, Maastricht University Medical Center, Maastricht, The Netherlands
| | - Gerard M J Bos
- Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center, Maastricht, The Netherlands; GROW-School for Oncology and Reproduction, Maastricht University, Maastricht, The Netherlands
| | - Lotte Wieten
- GROW-School for Oncology and Reproduction, Maastricht University, Maastricht, The Netherlands; Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands.
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19
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Ehlers FAI, Mahaweni NM, van de Waterweg Berends A, Saya T, Bos GMJ, Wieten L. Exploring the potential of combining IL-2-activated NK cells with an anti-PDL1 monoclonal antibody to target multiple myeloma-associated macrophages. Cancer Immunol Immunother 2023; 72:1789-1801. [PMID: 36656341 DOI: 10.1007/s00262-022-03365-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2022] [Accepted: 12/30/2022] [Indexed: 01/20/2023]
Abstract
Multiple myeloma (MM) is an incurable disease, characterized by malignant plasma cells in the bone marrow. MM growth is largely dependent on the tumor microenvironment (TME), consisting of complex cellular networks that shape a tumor-permissive environment. Within the TME, tumor-associated cells (TAC) comprise heterogeneous cell populations that collectively support immunosuppression. Reshaping the TME toward an immunostimulatory environment may enhance effectiveness of immunotherapies. Here, we investigated interactions between donor-derived natural killer (NK) cells and TAC, like tumor-associated macrophages (TAM) and M1 macrophages, and assessed whether anti-tumor effector functions of NK cells could be enhanced by an ADCC-triggering antibody targeting macrophages. Monocytes were polarized in vitro toward either M1 or TAM before co-culture with high-dose IL-2-activated NK cells. NK cell responses were assessed by measuring degranulation (CD107a) and IFN-γ production. We found that NK cells degranulated and produced IFN-γ upon interaction with both macrophage types. NK cell responses against PD-L1+ M1 macrophages could be further enhanced by Avelumab, an anti-PD-L1- and ADCC-inducing antibody. Additionally, NK cell responses were influenced by HLA class I, shown by stronger degranulation in NK cell subsets for which the corresponding HLA ligand was absent on the macrophage target cells (KIR-ligand mismatch) compared to degranulation in the presence of the HLA ligand (KIR-ligand match). Our results suggest that NK cells could, next to killing tumor cells, get activated upon interaction with TAC, like M1 macrophages and TAMs, and that NK cells combined with PD-L1 blocking antibodies with ADCC potential could, through IFN-γ secretion, promote a more immune-favorable TME.
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Affiliation(s)
- Femke A I Ehlers
- Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center+, Maastricht, The Netherlands.,Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center+, Maastricht, The Netherlands.,GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Niken M Mahaweni
- Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center+, Maastricht, The Netherlands.,Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center+, Maastricht, The Netherlands.,GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Annet van de Waterweg Berends
- Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center+, Maastricht, The Netherlands.,GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Thara Saya
- Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center+, Maastricht, The Netherlands.,GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Gerard M J Bos
- Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center+, Maastricht, The Netherlands.,GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Lotte Wieten
- Department of Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Center+, Maastricht, The Netherlands. .,GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands.
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20
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Halliez C, Ibrahim H, Otonkoski T, Mallone R. In vitro beta-cell killing models using immune cells and human pluripotent stem cell-derived islets: Challenges and opportunities. Front Endocrinol (Lausanne) 2023; 13:1076683. [PMID: 36726462 PMCID: PMC9885197 DOI: 10.3389/fendo.2022.1076683] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Accepted: 12/23/2022] [Indexed: 01/19/2023] Open
Abstract
Type 1 diabetes (T1D) is a disease of both autoimmunity and β-cells. The β-cells play an active role in their own demise by mounting defense mechanisms that are insufficient at best, and that can become even deleterious in the long term. This complex crosstalk is important to understanding the physiological defense mechanisms at play in healthy conditions, their alterations in the T1D setting, and therapeutic agents that may boost such mechanisms. Robust protocols to develop stem-cell-derived islets (SC-islets) from human pluripotent stem cells (hPSCs), and islet-reactive cytotoxic CD8+ T-cells from peripheral blood mononuclear cells offer unprecedented opportunities to study this crosstalk. Challenges to develop in vitro β-cell killing models include the cluster morphology of SC-islets, the relatively weak cytotoxicity of most autoimmune T-cells and the variable behavior of in vitro expanded CD8+ T-cells. These challenges may however be highly rewarding in light of the opportunities offered by such models. Herein, we discuss these opportunities including: the β-cell/immune crosstalk in an islet microenvironment; the features that make β-cells more sensitive to autoimmunity; therapeutic agents that may modulate β-cell vulnerability; and the possibility to perform analyses in an autologous setting, i.e., by generating T-cell effectors and SC-islets from the same donor.
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Affiliation(s)
- Clémentine Halliez
- Université Paris Cité, Institut Cochin, CNRS, INSERM, Paris, France
- Assistance Publique Hôpitaux de Paris, Service de Diabétologie et Immunologie Clinique, Cochin Hospital, Paris, France
| | - Hazem Ibrahim
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Timo Otonkoski
- Assistance Publique Hôpitaux de Paris, Service de Diabétologie et Immunologie Clinique, Cochin Hospital, Paris, France
- Department of Pediatrics, Helsinki University Hospital, Helsinki, Finland
| | - Roberto Mallone
- Université Paris Cité, Institut Cochin, CNRS, INSERM, Paris, France
- Assistance Publique Hôpitaux de Paris, Service de Diabétologie et Immunologie Clinique, Cochin Hospital, Paris, France
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Valenzuela-Vázquez L, Nuñez-Enriquez JC, Sánchez-Herrera J, Medina-Sanson A, Pérez-Saldivar ML, Jiménez-Hernández E, Martiín-Trejo JA, Del Campo-Martínez MDLÁ, Flores-Lujano J, Amador-Sánchez R, Mora-Ríos FG, Peñaloza-González JG, Duarte-Rodríguez DA, Torres-Nava JR, Espinosa-Elizondo RM, Cortés-Herrera B, Flores-Villegas LV, Merino-Pasaye LE, Almeida-Hernández C, Ramírez-Colorado R, Solís-Labastida KA, Medrano-López F, Pérez-Gómez JA, Velázquez-Aviña MM, Martínez-Ríos A, Aguilar-De los Santos A, Santillán-Juárez JD, Gurrola-Silva A, García-Velázquez AJ, Mata-Rocha M, Hernández-Echáurregui GA, Sepúlveda-Robles OA, Rosas-Vargas H, Mancilla-Herrera I, Jimenez-Morales S, Hidalgo-Miranda A, Martinez-Duncker I, Waight JD, Hance KW, Madauss KP, Mejía-Aranguré JM, Cruz-Munoz ME. NK cells with decreased expression of multiple activating receptors is a dominant phenotype in pediatric patients with acute lymphoblastic leukemia. Front Oncol 2022; 12:1023510. [PMID: 36419901 PMCID: PMC9677112 DOI: 10.3389/fonc.2022.1023510] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2022] [Accepted: 10/17/2022] [Indexed: 11/09/2022] Open
Abstract
NK cells have unique attributes to react towards cells undergoing malignant transformation or viral infection. This reactivity is regulated by activating or inhibitory germline encoded receptors. An impaired NK cell function may result from an aberrant expression of such receptors, a condition often seen in patients with hematological cancers. Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer worldwide and NK cells have emerged as crucial targets for developing immunotherapies. However, there are important gaps concerning the phenotype and behavior of NK cells during emergence of ALL. In this study we analyze the phenotype and function of NK cells from peripheral blood in pediatric patients with ALL at diagnosis. Our results showed that NK cells exhibited an altered phenotype highlighted by a significant reduction in the overall expression and percent representation of activating receptors compared to age-matched controls. No significant differences were found for the expression of inhibitory receptors. Moreover, NK cells with a concurrent reduced expression in various activating receptors, was the dominant phenotype among patients. An alteration in the relative frequencies of NK cells expressing NKG2A and CD57 within the mature NK cell pool was also observed. In addition, NK cells from patients displayed a significant reduction in the ability to sustain antibody-dependent cellular cytotoxicity (ADCC). Finally, an aberrant expression of activating receptors is associated with the phenomenon of leukemia during childhood.
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Affiliation(s)
- Lucero Valenzuela-Vázquez
- Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
- Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
| | - Juan Carlos Nuñez-Enriquez
- Unidad de Investigación Médica en Epidemiología Clínica, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Jacqueline Sánchez-Herrera
- Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
- Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
| | - Aurora Medina-Sanson
- Servicio de Oncología Pediátrica, Hospital Infantil de México, “Dr. Federico Gómez Sántos”, Secretaria de Salud, Ciudad de México, Mexico
| | - María Luisa Pérez-Saldivar
- Unidad de Investigación Médica en Epidemiología Clínica, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Elva Jiménez-Hernández
- Servicio de Hematología Pediátrica, Hospital General “Gaudencio González Garza”, Centro Médico Nacional (CMN) “La Raza”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Jorge Alfonso Martiín-Trejo
- Servicio de Hematología Pediátrica, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - María de Los Ángeles Del Campo-Martínez
- Servicio de Hematología Pediátrica, Hospital General “Gaudencio González Garza”, Centro Médico Nacional (CMN) “La Raza”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Janet Flores-Lujano
- Unidad de Investigación Médica en Epidemiología Clínica, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Raquel Amador-Sánchez
- Hospital General Regional No. 1 “Carlos McGregor Sánchez Navarro”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Félix Gustavo Mora-Ríos
- Departamento de Hematología, Hospital General Regional Ignacio Zaragoza del Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE), Mexico City, Mexico
| | | | - David Aldebarán Duarte-Rodríguez
- Unidad de Investigación Médica en Epidemiología Clínica, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - José Refugio Torres-Nava
- Servicio de Oncología, Hospital Pediátrico de Moctezuma, Secretaría de Salud de la Ciudad de México (CDMX), Mexico City, Mexico
| | | | - Beatriz Cortés-Herrera
- Servicio de Hematología Pediátrica, Hospital General de México, Secretaria de Salud (SS), Mexico City, Mexico
| | - Luz Victoria Flores-Villegas
- Servicio de Hematología Pediátrica, Centro Médico Nacional (CMN) “20 de Noviembre”, Instituto de Seguridad Social al Servicio de los Trabajadores del Estado (ISSSTE), Mexico City, Mexico
| | - Laura Elizabeth Merino-Pasaye
- Servicio de Hematología Pediátrica, Centro Médico Nacional (CMN) “20 de Noviembre”, Instituto de Seguridad Social al Servicio de los Trabajadores del Estado (ISSSTE), Mexico City, Mexico
| | - Carolina Almeida-Hernández
- Hospital General de Ecatepec “Las Américas”, Instituto de Salud del Estado de México (ISEM), Mexico City, Mexico
| | - Rosario Ramírez-Colorado
- Hospital Pediátrico La Villa, Secretaría de Salud de la Ciudad de México (SSCDMX), Mexico City, Mexico
| | - Karina Anastacia Solís-Labastida
- Servicio de Hematología Pediátrica, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Francisco Medrano-López
- Hospital General Regional (HGR) No. 72 “Dr. Vicente Santos Guajardo”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Jessica Arleet Pérez-Gómez
- Hospital General Regional (HGR) No. 72 “Dr. Vicente Santos Guajardo”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | | | - Annel Martínez-Ríos
- Departamento de Hematología, Hospital General Regional Ignacio Zaragoza del Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE), Mexico City, Mexico
| | | | - Jessica Denisse Santillán-Juárez
- Servicio de Hemato-oncología Pediátrica, Hospital Regional No. 1° de Octubre, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE), Mexico City, Mexico
| | - Alma Gurrola-Silva
- Hospital Regional Tipo B de Alta Especialidad Bicentenario de la Independencia, Instituto de Seguridad Social al Servicio de los Trabajadores del Estado, Mexico City, Mexico
| | - Alejandra Jimena García-Velázquez
- Servicio de Hemato-oncología Pediátrica, Hospital Regional No. 1° de Octubre, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE), Mexico City, Mexico
| | - Minerva Mata-Rocha
- Unidad de Investigación Médica en Epidemiología Clínica, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | | | - Omar Alejandro Sepúlveda-Robles
- Unidad de Investigación Médica en Genética Humana, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Haydeé Rosas-Vargas
- Unidad de Investigación Médica en Genética Humana, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Ismael Mancilla-Herrera
- Departamento de Infectología e Inmunología, Instituto Nacional de Perinatología, Mexico City, Mexico
| | - Silvia Jimenez-Morales
- Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
| | - Alfredo Hidalgo-Miranda
- Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
| | - Ivan Martinez-Duncker
- Centro de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
| | | | | | | | - Juan Manuel Mejía-Aranguré
- Unidad de Investigación Médica en Genética Humana, Unidad Médica de Alta Especialidad (UMAE) Hospital de Pediatría, Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
- Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
- Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico
- *Correspondence: Juan Manuel Mejía-Aranguré, ; Mario Ernesto Cruz-Munoz,
| | - Mario Ernesto Cruz-Munoz
- Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
- *Correspondence: Juan Manuel Mejía-Aranguré, ; Mario Ernesto Cruz-Munoz,
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22
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Huang L, He F, Wu B. Mechanism of effects of nickel or nickel compounds on intestinal mucosal barrier. CHEMOSPHERE 2022; 305:135429. [PMID: 35760131 DOI: 10.1016/j.chemosphere.2022.135429] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Revised: 06/17/2022] [Accepted: 06/18/2022] [Indexed: 06/15/2023]
Abstract
As an important metal in industry, national defense, and production, nickel widely exists in nature and is also a necessary trace element for human beings and animals. Nickel deficiency will affect the growth and development of animals, the contents of related active substances, enzymes and other essential elements in vivo. However, excessive nickel or longer nickel exposure can induce excessive free radicals (reactive oxygen species and reactive nitrogen) in the body, which can lead to a variety of cell damage, apoptosis and canceration, and ultimately pose negative effects on the health of the body. Among them, the intestinal tract, as the largest interface between the body and the external environment, greatly increases the contact probability between nickel or nickel compounds and the intestinal mucosal barrier, thus, the intestinal structure and function are also more vulnerable to nickel damage, leading to a series of related diseases such as enteritis. Therefore, this paper briefly analyzed the damage mechanism of nickel or its compounds to the intestinal tract from the perspective of four intestinal mucosal barriers: mechanical barrier, immune barrier, microbial barrier and chemical barrier, we hope to make a certain theoretical contribution to the further research and the prevention and treatment of nickel related diseases.
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Affiliation(s)
- Lijing Huang
- College of Life Sciences, China West Normal University, Nanchong, PR China
| | - Fang He
- College of Life Sciences, China West Normal University, Nanchong, PR China
| | - Bangyuan Wu
- Key Laboratory of Southwest China Wildlife Resources Conservation, Ministry of Education PR China, Nanchong, PR China; College of Life Sciences, China West Normal University, Nanchong, PR China.
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23
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Leukemia derived dendritic cell (DC leu) mediated immune response goes along with reduced (leukemia-specific) regulatory T-cells. Immunobiology 2022; 227:152237. [PMID: 35749805 DOI: 10.1016/j.imbio.2022.152237] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Revised: 05/18/2022] [Accepted: 06/07/2022] [Indexed: 12/17/2022]
Abstract
The blastmodulatory Kit-M, composed of granulocyte-macrophage colony-stimulating-factor (GM-CSF) and Prostaglandin E1 (PGE1), is known to convert myeloid leukaemic blasts (from AML patients) into leukaemia derived dendritic cells (DCleu), which activate immunoreactive cells to gain antileukemic/leukaemia-specific activity. In this study we had a special focus on the influence of Kit-M treated, DC/DCleu containing patients'whole blood (WB, n = 16) on the provision of immunosuppressive regulatory T-cells. We could confirm that Kit-M significantly increased frequencies of (mature) dendritic cells (DC) and DCleu from leukemic whole blood (WB) without induction of blast proliferation. After mixed lymphocyte culture (MLC) with patients' T-cells we confirmed that DCleu mediated leukemia-specific responses- going along with activated and leukemia-specific T- and NK-cells in an intracellular cytokine staining assay (ICS) and a degranulation assay (Deg)- resulted in an increased anti-leukemic cytotoxicity (Cytotoxicity Fluorolysis Assay = CTX). We could demonstrate that (leukemia-specific) CD4+ and CD8+ regulatory T-cell population (Treg) decreased significantly after MLC compared to controls. We found significant positive correlations of leukemia-specific CD3+CD4+ cells with frequencies of (mature) DCleu. Achieved anti-leukemic cytotoxicity correlated significantly positive with leukemia-specific CD3+CD8+ cells and significantly negatively with (leukemia-specific) Treg. In summary we demonstrate that immunesuppressive (leukemia-specific) regulatory T-cells are significantly downregulated after Kit-M triggered MLC- going along with a (reinstalled) antileukemic reactivity of the immune system (as demonstrated with functional assays ICS, Deg, CTX).
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24
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Jalali S, Harpur CM, Piers AT, Auladell M, Perriman L, Li S, An K, Anderson J, Berzins SP, Licciardi PV, Ashhurst TM, Konstantinov IE, Pellicci DG. A high-dimensional cytometry atlas of peripheral blood over the human life span. Immunol Cell Biol 2022; 100:805-821. [PMID: 36218032 PMCID: PMC9828744 DOI: 10.1111/imcb.12594] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Revised: 10/04/2022] [Accepted: 10/10/2022] [Indexed: 11/07/2022]
Abstract
Age can profoundly affect susceptibility to a broad range of human diseases. Children are more susceptible to some infectious diseases such as diphtheria and pertussis, while in others, such as coronavirus disease 2019 and hepatitis A, they are more protected compared with adults. One explanation is that the composition of the immune system is a major contributing factor to disease susceptibility and severity. While most studies of the human immune system have focused on adults, how the immune system changes after birth remains poorly understood. Here, using high-dimensional spectral flow cytometry and computational methods for data integration, we analyzed more than 50 populations of immune cells in the peripheral blood, generating an immune cell atlas that defines the healthy human immune system from birth up to 75 years of age. We focused our efforts on children under 18 years old, revealing major changes in immune cell populations after birth and in children of schooling age. Specifically, CD4+ T effector memory cells, Vδ2+ gamma delta (γδ)T cells, memory B cells, plasmablasts, CD11c+ B cells and CD16+ CD56bright natural killer (NK) cells peaked in children aged 5-9 years old, whereas frequencies of T helper 1, T helper 17, dendritic cells and CD16+ CD57+ CD56dim NK cells were highest in older children (10-18 years old). The frequency of mucosal-associated invariant T cells was low in the first several years of life and highest in adults between 19 and 30 years old. Late adulthood was associated with fewer mucosal-associated invariant T cells and Vδ2+ γδ T cells but with increased frequencies of memory subsets of B cells, CD4+ and CD8+ T cells and CD57+ NK cells. This human immune cell atlas provides a critical resource to understand changes to the immune system during life and provides a reference for investigating the immune system in the context of human disease. This work may also help guide future therapies that target specific populations of immune cells to protect at-risk populations.
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Affiliation(s)
- Sedigheh Jalali
- Murdoch Children's Research InstituteMelbourneVICAustralia,Department of PaediatricsUniversity of MelbourneMelbourneVICAustralia
| | | | - Adam T Piers
- Murdoch Children's Research InstituteMelbourneVICAustralia,Melbourne Centre for Cardiovascular Genomics and Regenerative MedicineMelbourneVICAustralia
| | - Maria Auladell
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and ImmunityUniversity of MelbourneMelbourneVICAustralia,Global Product Development Consulting for Infectious DiseasesPharmaceutical Product Development (PPD), Part of Thermo Fisher ScientificBennekomThe Netherlands
| | - Louis Perriman
- Murdoch Children's Research InstituteMelbourneVICAustralia,The Fiona Elsey Cancer Research InstituteBallaratVICAustralia,Federation UniversityBallaratVICAustralia
| | - Shuo Li
- Murdoch Children's Research InstituteMelbourneVICAustralia
| | - Kim An
- Murdoch Children's Research InstituteMelbourneVICAustralia,Melbourne Centre for Cardiovascular Genomics and Regenerative MedicineMelbourneVICAustralia
| | - Jeremy Anderson
- Murdoch Children's Research InstituteMelbourneVICAustralia,Department of PaediatricsUniversity of MelbourneMelbourneVICAustralia
| | - Stuart P Berzins
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and ImmunityUniversity of MelbourneMelbourneVICAustralia,The Fiona Elsey Cancer Research InstituteBallaratVICAustralia,Federation UniversityBallaratVICAustralia
| | - Paul V Licciardi
- Murdoch Children's Research InstituteMelbourneVICAustralia,Department of PaediatricsUniversity of MelbourneMelbourneVICAustralia
| | - Thomas M Ashhurst
- Sydney Cytometry Core Research FacilityThe University of Sydney and Centenary InstituteSydneyNSWAustralia,School of Medical Sciences, Faculty of Medicine and HealthThe University of SydneySydneyNSWAustralia
| | - Igor E Konstantinov
- Murdoch Children's Research InstituteMelbourneVICAustralia,Melbourne Centre for Cardiovascular Genomics and Regenerative MedicineMelbourneVICAustralia,Cardiothoracic SurgeryRoyal Children's HospitalMelbourneVICAustralia
| | - Daniel G Pellicci
- Murdoch Children's Research InstituteMelbourneVICAustralia,Department of PaediatricsUniversity of MelbourneMelbourneVICAustralia,Melbourne Centre for Cardiovascular Genomics and Regenerative MedicineMelbourneVICAustralia,Department of Microbiology and Immunology, Peter Doherty Institute for Infection and ImmunityUniversity of MelbourneMelbourneVICAustralia
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25
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Väyrynen JP, Haruki K, Lau MC, Väyrynen SA, Ugai T, Akimoto N, Zhong R, Zhao M, Dias Costa A, Borowsky J, Bell P, Takashima Y, Fujiyoshi K, Arima K, Kishikawa J, Shi SS, Twombly TS, Song M, Wu K, Chan AT, Zhang X, Fuchs CS, Meyerhardt JA, Giannakis M, Ogino S, Nowak JA. Spatial organization and prognostic significance of NK and NKT-like cells via multimarker analysis of the colorectal cancer microenvironment. Cancer Immunol Res 2021; 10:215-227. [DOI: 10.1158/2326-6066.cir-21-0772] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 11/24/2021] [Accepted: 12/20/2021] [Indexed: 11/16/2022]
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26
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Shui Y, Hu X, Hirano H, Kusano K, Tsukamoto H, Li M, Hasumi K, Guo WZ, Li XK. β-glucan from Aureobasidium pullulans augments the anti-tumor immune responses through activated tumor-associated dendritic cells. Int Immunopharmacol 2021; 101:108265. [PMID: 34715491 DOI: 10.1016/j.intimp.2021.108265] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 10/08/2021] [Accepted: 10/11/2021] [Indexed: 11/25/2022]
Abstract
Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells, capable of priming both naïve and memory T cells. Thus, tumor-resident DCs (tumor-associated DCs: TADCs) play a crucial role in the immune response against tumors. However, TADCs are also well known as a "double-edged sword" because an immunosuppressive environment, such as a tumor microenvironment, maintains the immature and tolerogenic properties of TADCs, resulting in the deterioration of the tumor. Therefore, it is essential to maintain and enhance the anti-tumoral activity of TADCs to aid tumor elimination. This study demonstrated the potential for tumor growth inhibition of Aureobasidium pullulan-derived β-glucan (AP-BG). Administration of AP-BG dramatically limited the development of different types of tumor cell lines transplanted into mice. Examination of the tumor-infiltrating leukocytes revealed that AP-BG caused high expression of co-stimulatory molecules on TADCs and enhanced the production of cytolytic granules as well as pro-inflammatory cytokines by the tumor-resident T cells. Furthermore, the syngeneic mixed lymphoid reaction assay and popliteal lymph node assay showed the significant ability of AP-BG to improve DCs' antigen-specific priming of T cells in vitro and in vivo. Taken together, β-glucan might be an immune-potentiating adjuvant for cancer treatment. This highly widely-used reagent will initiate a new way to activate DC-targeted cancer immune therapy.
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Affiliation(s)
- Yifang Shui
- Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; Division of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan; Department of Breast Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Xin Hu
- Division of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan
| | - Hiroshi Hirano
- Division of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan; Hasumi International Research Foundation, Tokyo, Japan
| | | | - Hirotake Tsukamoto
- Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; Division of Clinical Immunology and Cancer Immunotherapy, Center for Cancer Immunotherapy and Immunobiology, Graduate School of Medicine, Kyoto University, Japan
| | - Mengquan Li
- Department of Breast Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | | | - Wen-Zhi Guo
- Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
| | - Xiao-Kang Li
- Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; Division of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan.
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27
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Dannhauser D, Rossi D, Palatucci AT, Rubino V, Carriero F, Ruggiero G, Ripaldi M, Toriello M, Maisto G, Netti PA, Terrazzano G, Causa F. Non-invasive and label-free identification of human natural killer cell subclasses by biophysical single-cell features in microfluidic flow. LAB ON A CHIP 2021; 21:4144-4154. [PMID: 34515262 DOI: 10.1039/d1lc00651g] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Natural killer (NK) cells are indicated as favorite candidates for innovative therapeutic treatment and are divided into two subclasses: immature regulatory NK CD56bright and mature cytotoxic NK CD56dim. Therefore, the ability to discriminate CD56dim from CD56bright could be very useful because of their higher cytotoxicity. Nowadays, NK cell classification is routinely performed by cytometric analysis based on surface receptor expression. Here, we present an in-flow, label-free and non-invasive biophysical analysis of NK cells through a combination of light scattering and machine learning (ML) for NK cell subclass classification. In this respect, to identify relevant biophysical cell features, we stimulated NK cells with interleukine-15 inducing a subclass transition from CD56bright to CD56dim. We trained our ML algorithm with sorted NK cell subclasses (≥86% accuracy). Next, we applied our NK cell classification algorithm to cells stimulated over time, to investigate the transition of CD56bright to CD56dim and their biophysical feature changes. Finally, we tested our approach on several proband samples, highlighting the potential of our measurement approach. We show a label-free way for the robust identification of NK cell subclasses based on biophysical features, which can be applied in both cell biology and cell therapy.
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Affiliation(s)
- David Dannhauser
- Interdisciplinary Research Centre on Biomaterials (CRIB) and Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università degli Studi di Napoli "Federico II", Piazzale Tecchio 80, 80125 Naples, Italy.
| | - Domenico Rossi
- Center for Advanced Biomaterials for Healthcare@CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, 80125 Naples, Italy
| | - Anna Teresa Palatucci
- Dipartimento di Scienze (DiS), Università della Basilicata, Via dell'Ateneo Lucano 10, 85100 Potenza, Italy
| | - Valentina Rubino
- Dipartimento di Scienze Mediche Traslazionali, Università degli Studi di Napoli "Federico II", Naples, Italy
| | - Flavia Carriero
- Dipartimento di Scienze Mediche Traslazionali, Università degli Studi di Napoli "Federico II", Naples, Italy
| | - Giuseppina Ruggiero
- Dipartimento di Scienze Mediche Traslazionali, Università degli Studi di Napoli "Federico II", Naples, Italy
| | - Mimmo Ripaldi
- Dipartimento Oncologia AORN Santobono Pausilipon Hospital, Via Posillipo, 226, 80123, Naples, Italy
| | - Mario Toriello
- Dipartimento Oncologia AORN Santobono Pausilipon Hospital, Via Posillipo, 226, 80123, Naples, Italy
| | - Giovanna Maisto
- Dipartimento Oncologia AORN Santobono Pausilipon Hospital, Via Posillipo, 226, 80123, Naples, Italy
| | - Paolo Antonio Netti
- Interdisciplinary Research Centre on Biomaterials (CRIB) and Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università degli Studi di Napoli "Federico II", Piazzale Tecchio 80, 80125 Naples, Italy.
- Center for Advanced Biomaterials for Healthcare@CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, 80125 Naples, Italy
| | - Giuseppe Terrazzano
- Dipartimento di Scienze (DiS), Università della Basilicata, Via dell'Ateneo Lucano 10, 85100 Potenza, Italy
| | - Filippo Causa
- Interdisciplinary Research Centre on Biomaterials (CRIB) and Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università degli Studi di Napoli "Federico II", Piazzale Tecchio 80, 80125 Naples, Italy.
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Gonzalez VD, Huang YW, Delgado-Gonzalez A, Chen SY, Donoso K, Sachs K, Gentles AJ, Allard GM, Kolahi KS, Howitt BE, Porpiglia E, Fantl WJ. High-grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment. Cell Rep 2021; 36:109632. [PMID: 34469729 PMCID: PMC8546503 DOI: 10.1016/j.celrep.2021.109632] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Revised: 05/12/2021] [Accepted: 08/06/2021] [Indexed: 12/30/2022] Open
Abstract
Tubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3-CD16-) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy.
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MESH Headings
- Antineoplastic Agents/pharmacology
- Carboplatin/pharmacology
- Cell Line, Tumor
- Coculture Techniques
- Cytokines/metabolism
- Cytotoxicity, Immunologic
- Female
- Humans
- Immune Tolerance/drug effects
- Killer Cells, Natural/drug effects
- Killer Cells, Natural/immunology
- Killer Cells, Natural/metabolism
- Lymphocytes, Tumor-Infiltrating/drug effects
- Lymphocytes, Tumor-Infiltrating/immunology
- Lymphocytes, Tumor-Infiltrating/metabolism
- Neoplasms, Cystic, Mucinous, and Serous/drug therapy
- Neoplasms, Cystic, Mucinous, and Serous/immunology
- Neoplasms, Cystic, Mucinous, and Serous/metabolism
- Neoplasms, Cystic, Mucinous, and Serous/pathology
- Ovarian Neoplasms/drug therapy
- Ovarian Neoplasms/immunology
- Ovarian Neoplasms/metabolism
- Ovarian Neoplasms/pathology
- Phenotype
- Receptors, Natural Killer Cell/metabolism
- Tetraspanin 29/metabolism
- Trogocytosis
- Tumor Escape/drug effects
- Tumor Microenvironment/immunology
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Affiliation(s)
- Veronica D Gonzalez
- Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Ying-Wen Huang
- Department of Urology Stanford University School of Medicine, Stanford, CA 94305, USA
| | | | - Shih-Yu Chen
- Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Kenyi Donoso
- Department of Urology Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Karen Sachs
- Next Generation Analytics, Palo Alto, CA 94301, USA
| | - Andrew J Gentles
- Department of Medicine (Quantitative Sciences Unit, Biomedical Informatics) Biomedical Data Science, Stanford University School of Medicine, Stanford, CA 94305, USA; Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Grace M Allard
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Kevin S Kolahi
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Brooke E Howitt
- Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Ermelinda Porpiglia
- Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Wendy J Fantl
- Department of Urology Stanford University School of Medicine, Stanford, CA 94305, USA; Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA.
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Parent AV, Faleo G, Chavez J, Saxton M, Berrios DI, Kerper NR, Tang Q, Hebrok M. Selective deletion of human leukocyte antigens protects stem cell-derived islets from immune rejection. Cell Rep 2021; 36:109538. [PMID: 34407395 DOI: 10.1016/j.celrep.2021.109538] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Revised: 04/23/2021] [Accepted: 07/26/2021] [Indexed: 11/21/2022] Open
Abstract
Stem cell-based replacement therapies hold the promise to restore function of damaged or degenerated tissue such as the pancreatic islets in people with type 1 diabetes. Wide application of these therapies requires overcoming the fundamental roadblock of immune rejection. To address this issue, we use genetic engineering to create human pluripotent stem cells (hPSCs) in which the majority of the polymorphic human leukocyte antigens (HLAs), the main drivers of allogeneic rejection, are deleted. We retain the common HLA class I allele HLA-A2 and less polymorphic HLA-E/F/G to allow immune surveillance and inhibition of natural killer (NK) cells. We employ a combination of in vitro assays and humanized mouse models to demonstrate that these gene manipulations significantly reduce NK cell activity and T-cell-mediated alloimmune response against hPSC-derived islet cells. In summary, our approach produces hypoimmunogenic hPSCs that can be readily matched with recipients to avoid alloimmune rejection.
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Affiliation(s)
- Audrey V Parent
- Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
| | - Gaetano Faleo
- Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Jessica Chavez
- Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Michael Saxton
- Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
| | - David I Berrios
- Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Natanya R Kerper
- Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Qizhi Tang
- Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Matthias Hebrok
- Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
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Tonetti CR, de Souza-Araújo CN, Yoshida A, da Silva RF, Alves PCM, Mazzola TN, Derchain S, Fernandes LGR, Guimarães F. Ovarian Cancer-Associated Ascites Have High Proportions of Cytokine-Responsive CD56bright NK Cells. Cells 2021; 10:cells10071702. [PMID: 34359872 PMCID: PMC8306021 DOI: 10.3390/cells10071702] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2021] [Revised: 06/21/2021] [Accepted: 06/22/2021] [Indexed: 12/24/2022] Open
Abstract
Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.
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Affiliation(s)
- Cláudia Rodrigues Tonetti
- School of Medicine Sciences, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (C.R.T.); (C.N.d.S.-A.); (R.F.d.S.); (S.D.); (L.G.R.F.)
| | - Caroline Natânia de Souza-Araújo
- School of Medicine Sciences, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (C.R.T.); (C.N.d.S.-A.); (R.F.d.S.); (S.D.); (L.G.R.F.)
| | - Adriana Yoshida
- Centro de Atenção Integral à Saúde da Mulher (CAISM), Women’s Hospital José Aristodemo Pinotti, University of Campinas, Rua Alexander Fleming-101, Campinas CEP 13083-881, SP, Brazil;
| | - Rodrigo Fernandes da Silva
- School of Medicine Sciences, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (C.R.T.); (C.N.d.S.-A.); (R.F.d.S.); (S.D.); (L.G.R.F.)
| | - Paulo César Martins Alves
- Center for Investigation in Pediatrics, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (P.C.M.A.); (T.N.M.)
| | - Taís Nitsch Mazzola
- Center for Investigation in Pediatrics, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (P.C.M.A.); (T.N.M.)
| | - Sophie Derchain
- School of Medicine Sciences, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (C.R.T.); (C.N.d.S.-A.); (R.F.d.S.); (S.D.); (L.G.R.F.)
- Centro de Atenção Integral à Saúde da Mulher (CAISM), Women’s Hospital José Aristodemo Pinotti, University of Campinas, Rua Alexander Fleming-101, Campinas CEP 13083-881, SP, Brazil;
| | - Luís Gustavo Romani Fernandes
- School of Medicine Sciences, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (C.R.T.); (C.N.d.S.-A.); (R.F.d.S.); (S.D.); (L.G.R.F.)
| | - Fernando Guimarães
- School of Medicine Sciences, University of Campinas, Rua Tessália Vieira de Camargo-126, Campinas CEP 13083-887, SP, Brazil; (C.R.T.); (C.N.d.S.-A.); (R.F.d.S.); (S.D.); (L.G.R.F.)
- Centro de Atenção Integral à Saúde da Mulher (CAISM), Women’s Hospital José Aristodemo Pinotti, University of Campinas, Rua Alexander Fleming-101, Campinas CEP 13083-881, SP, Brazil;
- Correspondence: ; Tel.: +55-(19)-35219462
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ADCC-Inducing Antibody Trastuzumab and Selection of KIR-HLA Ligand Mismatched Donors Enhance the NK Cell Anti-Breast Cancer Response. Cancers (Basel) 2021; 13:cancers13133232. [PMID: 34203549 PMCID: PMC8268223 DOI: 10.3390/cancers13133232] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Revised: 06/07/2021] [Accepted: 06/23/2021] [Indexed: 11/17/2022] Open
Abstract
Simple Summary Natural killer (NK) cells are potent killers of tumor cells. Many tumors, including breast cancers, develop mechanisms to suppress anti-tumor immune responses, requiring the development of strategies to overcome suppression. Here, we tested a combination therapy that aims to (1) enhance NK cell activation and (2) reduce NK cell inhibition mediated by suppressive factors in tumors or in the tumor microenvironment. We cultured cell lines under hypoxia to mimic the tumor microenvironment or used patient-derived breast cancer cells that were primed by the patient’s tumor environment. Our results demonstrated that cytokine-activated NK cells remained active under hypoxia and that tumor-targeting antibodies enhanced the NK cell anti-breast cancer response. Moreover, we observed that NK cell suppression by inhibitory ligands on the tumor cells can be reduced by the selection of NK cell donors with NK receptors that are incompatible with these ligands. Collectively, we present two powerful strategies to enhance the NK cell responses against breast cancer. Abstract Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells’ full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.
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Harper J, Huot N, Micci L, Tharp G, King C, Rascle P, Shenvi N, Wang H, Galardi C, Upadhyay AA, Villinger F, Lifson J, Silvestri G, Easley K, Jacquelin B, Bosinger S, Müller-Trutwin M, Paiardini M. IL-21 and IFNα therapy rescues terminally differentiated NK cells and limits SIV reservoir in ART-treated macaques. Nat Commun 2021; 12:2866. [PMID: 34001890 PMCID: PMC8129202 DOI: 10.1038/s41467-021-23189-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Accepted: 04/08/2021] [Indexed: 12/13/2022] Open
Abstract
Unlike HIV infection, which progresses to AIDS absent suppressive anti-retroviral therapy, nonpathogenic infections in natural hosts, such African green monkeys, are characterized by a lack of gut microbial translocation and robust secondary lymphoid natural killer cell responses resulting in an absence of chronic inflammation and limited SIV dissemination in lymph node B-cell follicles. Here we report, using the pathogenic model of antiretroviral therapy-treated, SIV-infected rhesus macaques that sequential interleukin-21 and interferon alpha therapy generate terminally differentiated blood natural killer cells (NKG2a/clowCD16+) with potent human leukocyte antigen-E-restricted activity in response to SIV envelope peptides. This is in contrast to control macaques, where less differentiated, interferon gamma-producing natural killer cells predominate. The frequency and activity of terminally differentiated NKG2a/clowCD16+ natural killer cells correlates with a reduction of replication-competent SIV in lymph node during antiretroviral therapy and time to viral rebound following analytical treatment interruption. These data demonstrate that African green monkey-like natural killer cell differentiation profiles can be rescued in rhesus macaques to promote viral clearance in tissues.
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Affiliation(s)
- Justin Harper
- Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Nicolas Huot
- Institut Pasteur, Unité HIV, Inflammation et Persistance, Paris, France
| | - Luca Micci
- Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Gregory Tharp
- Nonhuman Primate Genomics Core, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Colin King
- Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Philippe Rascle
- Institut Pasteur, Unité HIV, Inflammation et Persistance, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, Paris, France
| | - Neeta Shenvi
- Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, GA, USA
| | - Hong Wang
- Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Cristin Galardi
- UNC HIV Cure Center and Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- HIV Discovery, ViiV Healthcare, Research Triangle Park, NC, USA
| | - Amit A Upadhyay
- Nonhuman Primate Genomics Core, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Francois Villinger
- Department of Biology, New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA, USA
| | - Jeffrey Lifson
- AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - Guido Silvestri
- Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
| | - Kirk Easley
- Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, GA, USA
| | | | - Steven Bosinger
- Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
- Nonhuman Primate Genomics Core, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
| | | | - Mirko Paiardini
- Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.
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Fathi E, Farahzadi R, Valipour B. Alginate/gelatin encapsulation promotes NK cells differentiation potential of bone marrow resident C-kit + hematopoietic stem cells. Int J Biol Macromol 2021; 177:317-327. [PMID: 33621568 DOI: 10.1016/j.ijbiomac.2021.02.131] [Citation(s) in RCA: 49] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 01/16/2021] [Accepted: 02/16/2021] [Indexed: 02/06/2023]
Abstract
The ability of natural killer (NK) cells to destroy cancerous cells with no prior sensitization has made them attractive candidates for cell therapy. The application of hydrogels must be notified as cell delivery vehicles in cell differentiation. The present study was conducted to investigate the effect of alginate-gelatin encapsulation on NK cell differentiation potential of C-kit+ cells. C-kit+ cells were differentiated to NK cells under both encapsulated and un-encapsulated conditions. Next, the cells were subjected to real-time polymerase chain reaction (PCR) and western blotting for the assessment of their telomere length and protein expressions, respectively. Afterward, culture medium was collected to measure cytokines levels. Thereafter, the differentiated NK cells were co-cultured with Molt-4 cells to investigate the potency of cell apoptosis by Annexin V/PI assay. A significant change was observed in the protein expression of Janus kinase/Signal transducers (JAK/STAT) pathway components. Additionally, the encapsulation caused an increase in the apoptosis of Molt-4 cells and telomere length of NK cells differentiated C-kit+ cells. Therefore, it can be concluded that the effects of encapsulation on NK cell's differentiation of C-kit+ cells could be resulted from the secreted cytokines of interleukin (IL)-2, IL-3, IL-7, and IL-12 as well as the increased telomere length.
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Affiliation(s)
- Ezzatollah Fathi
- Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran
| | - Raheleh Farahzadi
- Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
| | - Behnaz Valipour
- Department of Histopathology and Anatomy, Faculty of Medicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran
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Gossel LDH, Heim C, Pfeffermann LM, Moser LM, Bönig HB, Klingebiel TE, Bader P, Wels WS, Merker M, Rettinger E. Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor. Cancers (Basel) 2021; 13:cancers13061443. [PMID: 33809981 PMCID: PMC8004684 DOI: 10.3390/cancers13061443] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Revised: 03/14/2021] [Accepted: 03/18/2021] [Indexed: 12/11/2022] Open
Abstract
The dismal prognosis of pediatric and young adult patients with high-risk rhabdomyosarcoma (RMS) underscores the need for novel treatment options for this patient group. In previous studies, the tumor-associated surface antigen ERBB2 (HER2/neu) was identified as targetable in high-risk RMS. As a proof of concept, in this study, a novel treatment approach against RMS tumors using a genetically modified natural killer (NK)-92 cell line (NK-92/5.28.z) as an off-the-shelf ERBB2-chimeric antigen receptor (CAR)-engineered cell product was preclinically explored. In cytotoxicity assays, NK-92/5.28.z cells specifically recognized and efficiently eliminated RMS cell suspensions, tumor cell monolayers, and 3D tumor spheroids via the ERBB2-CAR even at effector-to-target ratios as low as 1:1. In contrast to unmodified parental NK-92 cells, which failed to lyse RMS cells, NK-92/5.28.z cells proliferated and became further activated through contact with ERBB2-positive tumor cells. Furthermore, high amounts of effector molecules, such as proinflammatory and antitumoral cytokines, were found in cocultures of NK-92/5.28.z cells with tumor cells. Taken together, our data suggest the enormous potential of this approach for improving the immunotherapy of treatment-resistant tumors, revealing the dual role of NK-92/5.28.z cells as CAR-targeted killers and modulators of endogenous adaptive immunity even in the inhibitory tumor microenvironment of high-risk RMS.
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Affiliation(s)
- Leonie D. H. Gossel
- Department for Children and Adolescents, Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, University Hospital Frankfurt, Goethe University, 60590 Frankfurt am Main, Germany; (L.D.H.G.); (C.H.); (L.M.M.); (P.B.); (M.M.)
| | - Catrin Heim
- Department for Children and Adolescents, Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, University Hospital Frankfurt, Goethe University, 60590 Frankfurt am Main, Germany; (L.D.H.G.); (C.H.); (L.M.M.); (P.B.); (M.M.)
| | - Lisa-Marie Pfeffermann
- Department of Cellular Therapeutics/Cell Processing, Institute for Transfusion Medicine and Immunohematology Frankfurt am Main, Goethe University Medical School, 60528 Frankfurt am Main, Germany; (L.-M.P.); (H.B.B.)
| | - Laura M. Moser
- Department for Children and Adolescents, Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, University Hospital Frankfurt, Goethe University, 60590 Frankfurt am Main, Germany; (L.D.H.G.); (C.H.); (L.M.M.); (P.B.); (M.M.)
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, 60590 Frankfurt am Main, Germany; (T.E.K.); (W.S.W.)
- Frankfurt Cancer Institute (FCI), 60596 Frankfurt am Main, Germany
- Universitäres Centrum für Tumorerkrankungen (UCT), 60590 Frankfurt am Main, Germany
| | - Halvard B. Bönig
- Department of Cellular Therapeutics/Cell Processing, Institute for Transfusion Medicine and Immunohematology Frankfurt am Main, Goethe University Medical School, 60528 Frankfurt am Main, Germany; (L.-M.P.); (H.B.B.)
- Department of Medicine, Division of Hematology, University of Washington, Seattle, WA 98198-7720, USA
| | - Thomas E. Klingebiel
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, 60590 Frankfurt am Main, Germany; (T.E.K.); (W.S.W.)
- Frankfurt Cancer Institute (FCI), 60596 Frankfurt am Main, Germany
- Universitäres Centrum für Tumorerkrankungen (UCT), 60590 Frankfurt am Main, Germany
- Department for Children and Adolescents, University Hospital Frankfurt, Goethe University, 60590 Frankfurt am Main, Germany
| | - Peter Bader
- Department for Children and Adolescents, Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, University Hospital Frankfurt, Goethe University, 60590 Frankfurt am Main, Germany; (L.D.H.G.); (C.H.); (L.M.M.); (P.B.); (M.M.)
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, 60590 Frankfurt am Main, Germany; (T.E.K.); (W.S.W.)
- Frankfurt Cancer Institute (FCI), 60596 Frankfurt am Main, Germany
- Universitäres Centrum für Tumorerkrankungen (UCT), 60590 Frankfurt am Main, Germany
| | - Winfried S. Wels
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, 60590 Frankfurt am Main, Germany; (T.E.K.); (W.S.W.)
- Frankfurt Cancer Institute (FCI), 60596 Frankfurt am Main, Germany
- Universitäres Centrum für Tumorerkrankungen (UCT), 60590 Frankfurt am Main, Germany
- Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, 60596 Frankfurt am Main, Germany
| | - Michael Merker
- Department for Children and Adolescents, Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, University Hospital Frankfurt, Goethe University, 60590 Frankfurt am Main, Germany; (L.D.H.G.); (C.H.); (L.M.M.); (P.B.); (M.M.)
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, 60590 Frankfurt am Main, Germany; (T.E.K.); (W.S.W.)
- Frankfurt Cancer Institute (FCI), 60596 Frankfurt am Main, Germany
- Universitäres Centrum für Tumorerkrankungen (UCT), 60590 Frankfurt am Main, Germany
| | - Eva Rettinger
- Department for Children and Adolescents, Division for Stem Cell Transplantation, Immunology and Intensive Care Medicine, University Hospital Frankfurt, Goethe University, 60590 Frankfurt am Main, Germany; (L.D.H.G.); (C.H.); (L.M.M.); (P.B.); (M.M.)
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, 60590 Frankfurt am Main, Germany; (T.E.K.); (W.S.W.)
- Frankfurt Cancer Institute (FCI), 60596 Frankfurt am Main, Germany
- Universitäres Centrum für Tumorerkrankungen (UCT), 60590 Frankfurt am Main, Germany
- Correspondence: ; Tel.: +49-(0)69-6301-80631; Fax: +49-(0)69-6301-4202
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Esteso G, Aguiló N, Julián E, Ashiru O, Ho MM, Martín C, Valés-Gómez M. Natural Killer Anti-Tumor Activity Can Be Achieved by In Vitro Incubation With Heat-Killed BCG. Front Immunol 2021; 12:622995. [PMID: 33708215 PMCID: PMC7940681 DOI: 10.3389/fimmu.2021.622995] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Accepted: 01/04/2021] [Indexed: 11/13/2022] Open
Abstract
Natural Killer cell receptors allow this heterogeneous immune population to efficiently fight both tumors and infection, so their use as immunotherapy agents is an active field of research. Cytokine activation, particularly by myeloid cell-derived IL15, can induce potent NK anti-tumor responses. While studying the mechanism of action of intravesical instillations of Bacille Calmette-Guérin (BCG) as therapy for patients with high risk non-muscle invasive bladder cancer, we showed that BCG can activate a cytotoxic CD56bright NK cell population which efficiently recognized bladder cancer cells. This pioneer immunotherapy provides an invaluable model to understand the role of different immune populations in tumor elimination. However, during the propagation of BCG worldwide a large number of genetically diverse BCG substrains developed. Here, we investigated the capacity of different BCG substrains to promote NK cell activation and confirmed that they were able to activate lymphocytes. Tice, Connaught and Moreau were the substrains with a stronger NK activation effect as measured by CD56 upregulation. Surprisingly, dead mycobacteria also stimulated PBMC cultures and we further demonstrate here that subcellular fractions of BCG-Tice, in the absence of live mycobacteria, could also induce an NK cell response. Lipids from BCG-Tice, but not from Mycobacterium bovis, stimulated NK cell activation and degranulation, whereas the aqueous fraction of either bacteria did not activate lymphocytes. However, delipidated BCG-Tice bacteria were able to activate effector cells (CD3+CD56+ and NK, CD3-CD56+). These data demonstrate that different components of mycobacteria can stimulate different immune subpopulations resulting in phenotypes suitable for cancer elimination.
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Affiliation(s)
- Gloria Esteso
- Department of Immunology and Oncology, Spanish National Centre for Biotechnology (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Nacho Aguiló
- Mycobacteria Genetics Group, University of Zaragoza Medical School, IIS Aragón, CIBERES, Zaragoza, Spain
| | - Esther Julián
- Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Omodele Ashiru
- Custom Solutions Department, Bacteriology Division, Medicines and Healthcare Products Regulatory Agency - National Institute for Biological Standards and Control (MHRA-NIBSC), Potters Bar, United Kingdom
| | - Mei M Ho
- Custom Solutions Department, Bacteriology Division, Medicines and Healthcare Products Regulatory Agency - National Institute for Biological Standards and Control (MHRA-NIBSC), Potters Bar, United Kingdom
| | - Carlos Martín
- Mycobacteria Genetics Group, University of Zaragoza Medical School, IIS Aragón, CIBERES, Zaragoza, Spain
| | - Mar Valés-Gómez
- Department of Immunology and Oncology, Spanish National Centre for Biotechnology (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
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36
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A CD8 + NK cell transcriptomic signature associated with clinical outcome in relapsing remitting multiple sclerosis. Nat Commun 2021; 12:635. [PMID: 33504809 PMCID: PMC7840761 DOI: 10.1038/s41467-020-20594-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Accepted: 11/24/2020] [Indexed: 02/07/2023] Open
Abstract
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) with the majority of cases characterised by relapsing/remitting (RRMS) attacks of neurologic dysfunction followed by variable resolution. Improving clinical outcomes in RRMS requires both a better understanding of the immunological mechanisms driving recurrent demyelination and better means of predicting future disease course to facilitate early targeted therapy. Here, we apply hypothesis-generating network transcriptomics to CD8+ cells isolated from patients in RRMS, identifying a signature reflecting expansion of a subset of CD8+ natural killer cells (NK8+) associated with favourable outcome. NK8+ are capable of regulating CD4+ T cell activation and proliferation in vitro, with reduced expression of HLA-G binding inhibitory receptors and consequent reduced sensitivity to HLA-G-mediated suppression. We identify surrogate markers of the NK8+ signature in peripheral blood leucocytes and validate their association with clinical outcome in an independent cohort, suggesting their measurement may facilitate early, targeted therapy in RRMS.
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Kim JM, Yi E, Cho H, Choi WS, Ko DH, Yoon DH, Hwang SH, Kim HS. Assessment of NK Cell Activity Based on NK Cell-Specific Receptor Synergy in Peripheral Blood Mononuclear Cells and Whole Blood. Int J Mol Sci 2020; 21:ijms21218112. [PMID: 33143099 PMCID: PMC7662667 DOI: 10.3390/ijms21218112] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2020] [Revised: 10/24/2020] [Accepted: 10/28/2020] [Indexed: 12/17/2022] Open
Abstract
Natural killer (NK) cells are cytotoxic innate lymphocytes endowed with a unique ability to kill a broad spectrum of cancer and virus-infected cells. Given their key contribution to diverse diseases, the measurement of NK cell activity (NKA) has been used to estimate disease prognosis or the effect of therapeutic treatment. Currently, NKA assays are primarily based on cumbersome procedures related to careful labeling and handling of target cells and/or NK cells, and they require a rapid isolation of peripheral blood mononuclear cells (PBMCs) which often necessitates a large amount of blood. Here, we developed an ELISA-based whole blood (WB) NKA assay involving engineered target cells (P815-ULBP1+CD48) providing defined and synergistic stimulation for NK cells via NKG2D and 2B4. WB collected from healthy donors (HDs) and patients with multiple myeloma (MM) was stimulated with P815-ULBP1+CD48 cells combined with IL-2. Thereafter, it utilized the serum concentrations of granzyme B and IFN-γ originating in NK cells as independent and complementary indicators of NKA. This WB NKA assay demonstrated that MM patients exhibit a significantly lower NKA than HDs following stimulation with P815-ULBP1+CD48 cells and had a good correlation with the commonly used flow cytometry-based PBMC NKA assay. Moreover, the use of P815-ULBP1+CD48 cells in relation to assessing the levels of NKG2D and 2B4 receptors on NK cells facilitated the mechanistic study and led to the identification of TGF-β1 as a potential mediator of compromised NKA in MM. Thus, our proposed WB NKA assay facilitates the reliable measurement of NKA and holds promise for further development as both a clinical and research tool.
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Affiliation(s)
- Jung Min Kim
- Asan Medical Center, Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea; (J.M.K.); (E.Y.); (W.S.C.)
| | - Eunbi Yi
- Asan Medical Center, Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea; (J.M.K.); (E.Y.); (W.S.C.)
| | - Hyungwoo Cho
- Asan Medical Center, Department of Oncology, University of Ulsan College of Medicine, Seoul 05505, Korea; (H.C.); (D.H.Y.)
| | - Woo Seon Choi
- Asan Medical Center, Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea; (J.M.K.); (E.Y.); (W.S.C.)
| | - Dae-Hyun Ko
- Asan Medical Center, Department of Laboratory Medicine, University of Ulsan College of Medicine, Seoul 05505, Korea;
| | - Dok Hyun Yoon
- Asan Medical Center, Department of Oncology, University of Ulsan College of Medicine, Seoul 05505, Korea; (H.C.); (D.H.Y.)
| | - Sang-Hyun Hwang
- Asan Medical Center, Department of Laboratory Medicine, University of Ulsan College of Medicine, Seoul 05505, Korea;
- Correspondence: (S.-H.H.); (H.S.K.)
| | - Hun Sik Kim
- Asan Medical Center, Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea; (J.M.K.); (E.Y.); (W.S.C.)
- Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505, Korea
- Correspondence: (S.-H.H.); (H.S.K.)
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38
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van der Heide SL, Xi Y, Upham JW. Natural Killer Cells and Host Defense Against Human Rhinoviruses Is Partially Dependent on Type I IFN Signaling. Front Cell Infect Microbiol 2020; 10:510619. [PMID: 33194777 PMCID: PMC7609819 DOI: 10.3389/fcimb.2020.510619] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Accepted: 10/02/2020] [Indexed: 12/12/2022] Open
Abstract
Rhinovirus (RV), the causative agent of the common cold, causes only mild upper respiratory tract infections in healthy individuals, but can cause longer lasting and more severe pulmonary infections in people with chronic lung diseases and in the setting of immune suppression or immune deficiency. RV-infected lung structural cells release type I interferon (IFN-I), initiating the immune response, leading to protection against viruses in conjunction with migratory immune cells. However, IFN-I release is deficient in some people with asthma. Innate immune cells, such as natural killer (NK) cells, are proposed to play major roles in the control of viral infections, and may contribute to exacerbations of chronic lung diseases, such as asthma. In this study, we characterized the NK cell response to RV infection using an in vitro model of infection in healthy individuals, and determined the extent to which IFN-I signaling mediates this response. The results indicate that RV stimulation in vitro induces NK cell activation in healthy donors, leading to degranulation and the release of cytotoxic mediators and cytokines. IFN-I signaling was partly responsible for NK cell activation and functional responses to RV. Overall, our findings suggest the involvement of NK cells in the control of RV infection in healthy individuals. Further understanding of NK cell regulation may deepen our understanding of the mechanisms that contribute to susceptibility to RV infections in asthma and other chronic lung diseases.
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Affiliation(s)
- Saskia L van der Heide
- Lung and Allergy Research Centre, Diamantina Institute, The University of Queensland, Woolloongabba, QLD, Australia
| | - Yang Xi
- Lung and Allergy Research Centre, Diamantina Institute, The University of Queensland, Woolloongabba, QLD, Australia
| | - John W Upham
- Lung and Allergy Research Centre, Diamantina Institute, The University of Queensland, Woolloongabba, QLD, Australia.,Department of Respiratory Medicine, Princess Alexandra Hospital, Brisbane, QLD, Australia
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Abdel-Latif M, Youness RA. Why natural killer cells in triple negative breast cancer? World J Clin Oncol 2020; 11:464-476. [PMID: 32821652 PMCID: PMC7407924 DOI: 10.5306/wjco.v11.i7.464] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Revised: 04/28/2020] [Accepted: 05/29/2020] [Indexed: 02/06/2023] Open
Abstract
The triple-negative subtype of breast cancer (TNBC) has the bleakest prognosis, owing to its lack of either hormone receptor as well as human epidermal growth factor receptor 2. Henceforth, immunotherapy has emerged as the front-runner for TNBC treatment, which avoids potentially damaging chemotherapeutics. However, despite its documented association with aggressive side effects and developed resistance, immune checkpoint blockade continues to dominate the TNBC immunotherapy scene. These immune checkpoint blockade drawbacks necessitate the exploration of other immunotherapeutic methods that would expand options for TNBC patients. One such method is the exploitation and recruitment of natural killer cells, which by harnessing the innate rather than adaptive immune system could potentially circumvent the downsides of immune checkpoint blockade. In this review, the authors will elucidate the advantageousness of natural killer cell-based immuno-oncology in TNBC as well as demonstrate the need to more extensively research such therapies in the future.
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Affiliation(s)
- Mustafa Abdel-Latif
- Biotechnology Program, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
| | - Rana Ahmed Youness
- Pharmaceutical Biology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
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40
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Freitas Monteiro M, Papaserafeim M, Réal A, Puga Yung GL, Seebach JD. Anti-CD20 rituximab IgG1, IgG3, and IgG4 but not IgG2 subclass trigger Ca 2+ mobilization and cytotoxicity in human NK cells. J Leukoc Biol 2020; 108:1409-1423. [PMID: 32620047 DOI: 10.1002/jlb.5ma0620-039r] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 06/11/2020] [Accepted: 06/16/2020] [Indexed: 12/12/2022] Open
Abstract
NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc-gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses and FCGR3A V158F single nucleotide polymorphism (SNP) on Ca2+ signaling and NK cell-mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with different FCGR3A SNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca2+ signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low-affinity (FF) variant for the FCGR3A V158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high-affinity (VV) and VF SNP variants was two- to threefold higher. Variations of FCGR3A SNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca2+ signaling and degranulation; however, ADCC was somewhat weaker in donors with the low-affinity FF variation. In conclusion, this is the first study correlating Ca2+ signaling and NK cell-mediated ADCC triggered by the four IgG subclasses with the FCGR3A V158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with FcγRIIIA/CD16a but no major impact of FCGR3A SNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy.
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Affiliation(s)
- Marta Freitas Monteiro
- Division of Immunology and Allergology, Department of Medicine, University Hospitals and Medical Faculty, Geneva, Switzerland
| | - Maria Papaserafeim
- Division of Immunology and Allergology, Department of Medicine, University Hospitals and Medical Faculty, Geneva, Switzerland
| | - Aline Réal
- Division of Immunology and Allergology, Department of Medicine, University Hospitals and Medical Faculty, Geneva, Switzerland
| | - Gisella L Puga Yung
- Division of Immunology and Allergology, Department of Medicine, University Hospitals and Medical Faculty, Geneva, Switzerland
| | - Jörg D Seebach
- Division of Immunology and Allergology, Department of Medicine, University Hospitals and Medical Faculty, Geneva, Switzerland
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41
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Improving Immunotherapy Against B-Cell Malignancies Using γδ T-Cell-specific Stimulation and Therapeutic Monoclonal Antibodies. J Immunother 2020; 42:331-344. [PMID: 31318724 DOI: 10.1097/cji.0000000000000289] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Tumor antigen-targeting monoclonal antibodies (mAbs) are an important element of current cancer therapies. Some of these therapeutic mAbs enable antibody-dependent cell mediated cytotoxicity (ADCC) against tumor cells. However, cancer-related functional impairment of immune effector cells may limit the clinical efficacy of antibody treatments. We reckoned that combining mAbs with cell-based immunotherapies would provide a clinically relevant synergism and benefit for cancer patients. Here, we focus on γδ T cells, as earlier studies demonstrated that γδ T-cell-based therapies are safe and promising for several types of malignancies. Similar to natural killer cells, their antitumor effects can be enhanced using antibodies, and they could, therefore, become a versatile effector cell platform for use with a variety of licensed therapeutic mAbs against cancer. In this study, we explore the potential of a combination therapy of activated γδ T cells with rituximab and the more recently developed mAbs (obinutuzumab and daratumumab) in different B-cell malignancies in vitro. Obinutuzumab outperformed the other mAbs with regard to direct target cell lysis and ADCC by γδ T cells in several CD20 cell lines and primary lymphoma specimens. We demonstrate that comparatively few CD16 γδ T cells are sufficient to mediate a strong ADCC. Using Fc-receptor-positive B-cell lymphomas as target cells, ADCC cannot be blocked by high concentrations of immunoglobulins or anti-CD16 antibodies, but both substances can promote cell mediated target cell lysis. This study expands on earlier reports on the therapeutic potential of distinctive tumor antigen-targeting mAbs and facilitates the understanding of the mechanism and potential of ADCC by γδ T-cell subsets.
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42
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Zamame Ramirez JA, Romagnoli GG, Falasco BF, Gorgulho CM, Sanzochi Fogolin C, Dos Santos DC, Junior JPA, Lotze MT, Ureshino RP, Kaneno R. Blocking drug-induced autophagy with chloroquine in HCT-116 colon cancer cells enhances DC maturation and T cell responses induced by tumor cell lysate. Int Immunopharmacol 2020; 84:106495. [PMID: 32298965 PMCID: PMC7152898 DOI: 10.1016/j.intimp.2020.106495] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 04/03/2020] [Accepted: 04/06/2020] [Indexed: 01/24/2023]
Abstract
Autophagy is an important mechanism for tumor escape, allowing tumor cells to recover from the damage induced by chemotherapy, radiation therapy, and immunotherapy and contributing to the development of resistance. The pharmacological inhibition of autophagy contributes to increase the efficacy of antineoplastic agents. Exposing tumor cells to low concentrations of select autophagy-inducing antineoplastic agents increases their immunogenicity and enhances their ability to stimulate dendritic cell (DC) maturation. We tested whether the application of an autophagy-inhibiting agent, chloroquine (CQ), in combination with low concentrations of 5-fluorouracil (5-FU) increases the ability of tumor cells to induce DC maturation. DCs sensitized with the lysate of HCT-116 cells previously exposed to such a combination enhanced the DC maturation/activation ability. These matured DCs also increased the allogeneic responsiveness of both CD4+ and CD8+ T cells, which showed a greater proliferative response than those from DCs sensitized with control lysates. The T cells expanded in such cocultures were CD69+ and PD-1- and produced higher levels of IFN-γ and lower levels of IL-10, consistent with the preferential activation of Th1 cells. Cocultures of autologous DCs and lymphocytes improved the generation of cytotoxic T lymphocytes, as assessed by the expression of CD107a, perforin, and granzyme B. The drug combination increased the expression of genes related to the CEACAM family (BECN1, ATGs, MAPLC3B, ULK1, SQSTM1) and tumor suppressors (PCBP1). Furthermore, the decreased expression of genes related to metastasis and tumor progression (BNIP3, BNIP3L, FOSL2, HES1, LAMB3, LOXL2, NDRG1, P4HA1, PIK3R2) was noted. The combination of 5-FU and CQ increases the ability of tumor cells to drive DC maturation and enhances the ability of DCs to stimulate T cell responses.
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Affiliation(s)
- Jofer Andree Zamame Ramirez
- São Paulo State University - UNESP, Department of Chemical and Biological Sciences, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil; São Paulo State University - UNESP, Department of Pathology, School of Medicine of Botucatu, Botucatu, SP, Brazil
| | - Graziela Gorete Romagnoli
- São Paulo State University - UNESP, Department of Chemical and Biological Sciences, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil; São Paulo State University - UNESP, Department of Pathology, School of Medicine of Botucatu, Botucatu, SP, Brazil
| | - Bianca Francisco Falasco
- São Paulo State University - UNESP, Department of Chemical and Biological Sciences, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil
| | - Carolina Mendonça Gorgulho
- São Paulo State University - UNESP, Department of Chemical and Biological Sciences, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil; São Paulo State University - UNESP, Department of Pathology, School of Medicine of Botucatu, Botucatu, SP, Brazil
| | - Carla Sanzochi Fogolin
- São Paulo State University - UNESP, Department of Chemical and Biological Sciences, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil
| | - Daniela Carvalho Dos Santos
- São Paulo State University - UNESP, Center for Electron Microscopy, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil
| | - João Pessoa Araújo Junior
- São Paulo State University - UNESP, Department of Chemical and Biological Sciences, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil
| | - Michael Thomas Lotze
- Department of Immunology, University of Pittsburgh, Pittsburgh, PA, United States
| | | | - Ramon Kaneno
- São Paulo State University - UNESP, Department of Chemical and Biological Sciences, Institute of Biosciences of Botucatu, Botucatu, SP, Brazil.
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Li M, Xiong Y, Li M, Zhang W, Liu J, Zhang Y, Xiong S, Zou C, Liang B, Lu M, Yang D, Peng C, Zheng X. Depletion but Activation of CD56 dimCD16 + NK Cells in Acute Infection with Severe Fever with Thrombocytopenia Syndrome Virus. Virol Sin 2020; 35:588-598. [PMID: 32430872 DOI: 10.1007/s12250-020-00224-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2019] [Accepted: 02/28/2020] [Indexed: 10/24/2022] Open
Abstract
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high mortality (12%-30%). The mechanism by which the SFTS bunyavirus (SFTSV) causes severe illness remains unclear. To evaluate the phenotypic and functional characteristics of the NK cell subsets in SFTS patients, twenty-nine SFTS patients were sequentially sampled from admission until recovery. Phenotypic and functional characteristics of NK cell subsets in circulating blood were analysed via flow cytometry. Then, correlations between NK cell subset frequencies and the SFTS index (SFTSI) were evaluated in all SFTS patients (15 mild, 14 severe) upon admission. The frequencies of CD56dimCD16+ NK cells were greatly decreased in early SFTSV infection and were negatively correlated with disease severity. Additionally, higher Ki-67 and granzyme B expression and relatively lower NKG2A expression in CD56dimCD16+ NK cells were observed in acute infection. Moreover, the effector function of CD56dim NK cells was increased in the acute phase compared with the recovery phase in nine severe SFTS patients. Additionally, interleukin (IL)-15, interferon (IFN)-α, IL-18 and IFN-γ secretion was markedly increased during early infection. Collectively, despite depletion of CD56dimCD16+ NK cells, activation and functional enhancement of CD56dimCD16+ NK cells were still observed, suggesting their involvement in defence against early SFTSV infection.
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Affiliation(s)
- Mengmeng Li
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.,Department of Gastroenterology, Henan Provincial People's Hospital, People's Hospital of Zhengzhou University, Zhengzhou, 450003, China
| | - Yan Xiong
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.,Department of Gastroenterology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021, China
| | - Mingyue Li
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Wenjing Zhang
- Department of Paediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430032, China
| | - Jia Liu
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Yanfang Zhang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
| | - Shue Xiong
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Congcong Zou
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Boyun Liang
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Mengji Lu
- Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Essen, 45122, Germany
| | - Dongliang Yang
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Cheng Peng
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
| | - Xin Zheng
- Department of Infectious Diseases, Institute of Infection and Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
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Various Stages of Immune Synapse Formation Are Differently Dependent on the Strength of the TCR Stimulus. Int J Mol Sci 2020; 21:ijms21072475. [PMID: 32252488 PMCID: PMC7177831 DOI: 10.3390/ijms21072475] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 03/23/2020] [Accepted: 03/31/2020] [Indexed: 12/13/2022] Open
Abstract
Cytotoxic T lymphocytes (CTL) are key players of the adaptive immune system that target tumors and infected cells. A central step to that is the formation of a cell–cell contact zone between the CTL and its target called an immune synapse (IS). Here, we investigate the influence of the initial T cell receptor (TCR) trigger of a cytolytic IS on the distinct steps leading to cytotoxic granule (CG) exocytosis. We stimulated primary CTLs from mouse using lipid bilayers with varying anti-CD3 but constant ICAM concentrations. We fluorescently labeled molecular markers of distinct IS zones such as actin, CD3, granzyme B, and Synaptobrevin2 in CTLs and imaged cytolytic IS formation by total internal reflection fluorescence microscopy (TIRFM). We found that an intermediate anti-CD3 concentration of 10 µg/mL induces the fastest adhesion of CTLs to the bilayers and results in maximal CG fusion efficiency. The latency of actin ring formation, dwell time, and maximum surface area at the IS exhibit different dependencies on the stimulatory anti-CD3 concentrations. The number and surface area of CD3 clusters at the IS seem to show a different dependency to the TCR trigger when compared to their dwell time. Finally, the mode of full CG exocytosis appears to be independent of the TCR trigger.
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45
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Schilbach K, Krickeberg N, Kaißer C, Mingram S, Kind J, Siegers GM, Hashimoto H. Suppressive activity of Vδ2 + γδ T cells on αβ T cells is licensed by TCR signaling and correlates with signal strength. Cancer Immunol Immunother 2020; 69:593-610. [PMID: 31982940 PMCID: PMC7113223 DOI: 10.1007/s00262-019-02469-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2019] [Accepted: 12/30/2019] [Indexed: 12/14/2022]
Abstract
Despite recent progress in the understanding of γδ T cells' roles and functions, their interaction with αβ T cells still remains to be elucidated. In this study, we sought to clarify what precisely endows peripheral Vδ2+ T cells with immunosuppressive function on autologous αβ T cells. We found that negatively freshly isolated Vδ2+ T cells do not exhibit suppressive behavior, even after stimulation with IL-12/IL-18/IL-15 or the sheer contact with butyrophilin-3A1-expressing tumor cell lines (U251 or SK-Mel-28). On the other hand, Vδ2+ T cells positively isolated through TCR crosslinking or after prolonged stimulation with isopentenyl pyrophosphate (IPP) mediate strong inhibitory effects on αβ T cell proliferation. Stimulation with IPP in the presence of IL-15 induces the most robust suppressive phenotype of Vδ2+ T cells. This indicates that Vδ2+ T cells' suppressive activity is dependent on a TCR signal and that the degree of suppression correlates with its strength. Vδ2+ T cell immunosuppression does not correlate with their Foxp3 expression but rather with their PD-L1 protein expression, evidenced by the massive reduction of suppressive activity when using a blocking antibody. In conclusion, pharmacologic stimulation of Vδ2+ T cells via the Vδ2 TCR for activation and expansion induces Vδ2+ T cells' potent killer activity while simultaneously licensing them to suppress αβ T cell responses. Taken together, the study is a further step to understand-in more detail-the suppressive activity of Vδ2+ γδ T cells.
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MESH Headings
- Apoptosis/drug effects
- Apoptosis/immunology
- B7-H1 Antigen/genetics
- B7-H1 Antigen/immunology
- B7-H1 Antigen/metabolism
- Cell Line, Tumor
- Cell Proliferation/drug effects
- Cells, Cultured
- Gene Expression/drug effects
- Gene Expression/immunology
- Hemiterpenes/pharmacology
- Humans
- Immune Tolerance/drug effects
- Immune Tolerance/genetics
- Immune Tolerance/immunology
- Interleukin-15/pharmacology
- Lymphocyte Activation/drug effects
- Lymphocyte Activation/immunology
- Organophosphorus Compounds/pharmacology
- Receptors, Antigen, T-Cell/immunology
- Receptors, Antigen, T-Cell/metabolism
- Receptors, Antigen, T-Cell, gamma-delta/immunology
- Receptors, Antigen, T-Cell, gamma-delta/metabolism
- Signal Transduction/drug effects
- Signal Transduction/immunology
- T-Lymphocyte Subsets/drug effects
- T-Lymphocyte Subsets/immunology
- T-Lymphocyte Subsets/metabolism
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Affiliation(s)
- Karin Schilbach
- Department of Pediatric Hematology and Oncology, University Children's Hospital Tübingen, Hoppe-Seyler Street 1, 72076, Tübingen, Germany.
| | - Naomi Krickeberg
- Department of Pediatric Hematology and Oncology, University Children's Hospital Tübingen, Hoppe-Seyler Street 1, 72076, Tübingen, Germany
| | - Carlotta Kaißer
- Department of Pediatric Hematology and Oncology, University Children's Hospital Tübingen, Hoppe-Seyler Street 1, 72076, Tübingen, Germany
| | - Simon Mingram
- Department of Pediatric Hematology and Oncology, University Children's Hospital Tübingen, Hoppe-Seyler Street 1, 72076, Tübingen, Germany
| | - Janika Kind
- Department of Pediatric Hematology and Oncology, University Children's Hospital Tübingen, Hoppe-Seyler Street 1, 72076, Tübingen, Germany
| | | | - Hisayoshi Hashimoto
- Department of Pediatric Hematology and Oncology, University Children's Hospital Tübingen, Hoppe-Seyler Street 1, 72076, Tübingen, Germany
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46
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Ortega-Rodríguez AC, Marín-Jáuregui LS, Martínez-Shio E, Hernández Castro B, González-Amaro R, Escobedo-Uribe CD, Monsiváis-Urenda AE. Altered NK cell receptor repertoire and function of natural killer cells in patients with acute myocardial infarction: A three-month follow-up study. Immunobiology 2020; 225:151909. [PMID: 32051096 DOI: 10.1016/j.imbio.2020.151909] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Accepted: 01/29/2020] [Indexed: 01/09/2023]
Abstract
NK cells are important in the onset of acute myocardial infarction (AMI) by their ability to secrete IFN-γ and other inflammatory cytokines. They also participate in regulating pathological cardiac remodeling after myocardial infarction. Mechanisms of regulation, however, are incompletely understood. Herein, the aim of this study is to explore the possible association between the expression pattern of different NK cell receptors (phenotype), as well as the cytotoxic function of NK cells from AMI patients with their myocardial function after three months follow-up. We analyzed the phenotype and function of both CD56dimCD16+ and CD56brightCD16- NK cells from twenty-one patients within the first 72 h after ST-elevation AMI and three-month follow-up, as well as fifteen healthy controls. Clinical characteristics and ventricular function determined by echocardiography were also evaluated. NK cells from AMI patients showed an activated phenotype, characterized by high TNF-α production and low percentages of the activating receptor NKG2D. Interestingly, AMI patients display higher levels of circulating IL-10+ NK cells. Three-month follow-up showed that NK cells exhibit a diminished cytotoxic function. These data show that NK cells may have a role mediating myocardial remodeling by regulating the inflammatory response, mainly by the production of IL-10. We also propose that NKG2D may have a role in the onset of the inflammatory response immediately after AMI. The precise regulation of NK cells function may represent an important step in recovery of myocardial function.
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Affiliation(s)
- Alma Celeste Ortega-Rodríguez
- Medicina Molecular y Traslacional, Centro de Investigación en Ciencias de la Salud y Biomedicina, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico
| | - Laura Sherell Marín-Jáuregui
- Medicina Molecular y Traslacional, Centro de Investigación en Ciencias de la Salud y Biomedicina, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico
| | - Elena Martínez-Shio
- Medicina Molecular y Traslacional, Centro de Investigación en Ciencias de la Salud y Biomedicina, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico
| | - Berenice Hernández Castro
- Medicina Molecular y Traslacional, Centro de Investigación en Ciencias de la Salud y Biomedicina, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico
| | - Roberto González-Amaro
- Medicina Molecular y Traslacional, Centro de Investigación en Ciencias de la Salud y Biomedicina, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico
| | | | - Adriana E Monsiváis-Urenda
- Medicina Molecular y Traslacional, Centro de Investigación en Ciencias de la Salud y Biomedicina, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico.
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47
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Valenzuela-Vazquez L, Núñez-Enríquez JC, Sánchez-Herrera J, Jiménez-Hernández E, Martín-Trejo JA, Espinoza-Hernández LE, Medina-Sanson A, Flores-Villegas LV, Peñaloza-González JG, Refugio Torres-Nava J, Espinosa-Elizondo RM, Amador-Sánchez R, Santillán-Juárez JD, Flores-Lujano J, Pérez-Saldívar ML, García-López LR, Castañeda-Echevarría A, Rodríguez-Leyva F, Rosas-Vargas H, Mata-Rocha M, Duarte-Rodríguez DA, Sepúlveda-Robles OA, Mancilla-Herrera I, Mejía-Aranguré JM, Cruz-Munoz ME. Functional characterization of NK cells in Mexican pediatric patients with acute lymphoblastic leukemia: Report from the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia. PLoS One 2020; 15:e0227314. [PMID: 31951638 PMCID: PMC6968843 DOI: 10.1371/journal.pone.0227314] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2019] [Accepted: 12/16/2019] [Indexed: 12/16/2022] Open
Abstract
Acute lymphoblastic leukemia (ALL) is the most common cancer in children around the globe. Mexico City has one of the highest incidence rates of childhood leukemia worldwide with 49.5 cases per million children under the age of 15 which is similar to that reported for Hispanic populations living in the United States. In addition, it has been noted a dismal prognosis in Mexican and Hispanic ALL pediatric population. Although ALL, like cancer in general, has its origins in endogenous, exogenous, and genetic factors, several studies have shown that the immune system also plays a deterministic role in cancer development. Among various elements of the immune system, T lymphocytes and NK cells seem to dominate the immune response against leukemia. The aim of the present study was to perform a phenotypic and functional characterization of NK cells in ALL Mexican children at the moment of diagnosis and before treatment initiation. A case-control study was conducted by the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia (MIGICCL). 41 cases were incident ALL children younger than 17 years old and residents of Mexico City. 14 controls were children without leukemia, matched by age and sex with cases. NK cell function was evaluated by degranulation assays towards K562 cells and SLAM-associated protein (SAP) expression was measured by intracellular staining. All assays were performed using peripheral blood mononuclear cells from controls and patients. The results indicate that NK mediated cytotoxicity, measured by CD107a degranulation assays in response to K562 cells, was reduced in ALL patients compared to controls. Interestingly, an impaired NK cell killing of target cells was not equally distributed among ALL patients. In contrast to patients classified as high-risk, standard-risk patients did not display a significant reduction in NK cell-mediated cytotoxicity. Moreover, patients presenting a leukocyte count ≥ 50,000xmm3 displayed a reduction in NK-cell mediated cytotoxicity and a reduction in SAP expression, indicating a positive correlation between a reduced SAP expression and an impaired NK cell-mediated citotoxicity. In the present study it was observed that unlike patients with standard-risk, NK cells from children presenting high-risk ALL, harbor an impaired cytotoxicity towards K562 at diagnosis. In addition, NK cell function was observed to be compromised in patients with a leukocyte count ≥50,000xmm3, where also it was noticed a decreased expression of SAP compared to patients with a leukocyte count <50,000xmm3. These data indicate NK cell-mediated cytotoxicity is not equally affected in ALL patients, nevertheless a positive correlation between low SAP expression and decreased NK cell-mediated cytotoxicity was observed in ALL patients with a leukocyte count ≥50,000xmm3. Finally, an abnormal NK cell-mediated cytotoxicity may represent a prognostic factor for high-risk acute lymphoblastic leukemia.
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Affiliation(s)
| | - Juan Carlos Núñez-Enríquez
- Unidad de Investigación Médica en Epidemiología Clínica, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | | | - Elva Jiménez-Hernández
- Servicio de Hematología Pediátrica, Hospital General “Gaudencio González Garza”, Centro Médico Nacional (CMN) "La Raza", IMSS, Mexico City, Mexico
| | - Jorge Alfonso Martín-Trejo
- Servicio de Hematología Pediátrica, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Laura Eugenia Espinoza-Hernández
- Servicio de Hematología Pediátrica, Hospital General “Gaudencio González Garza”, Centro Médico Nacional (CMN) "La Raza", IMSS, Mexico City, Mexico
| | - Aurora Medina-Sanson
- Servicio de Hemato-Oncologia, Hospital Infantil de México Federico Gómez, Secretaria de Salud (SS), Mexico City, Mexico
| | - Luz Victoria Flores-Villegas
- Servicio de Hematología Pediátrica, Centro Médico Nacional (CMN) “20 de Noviembre”, Instituto de Seguridad Social al Servicio de los Trabajadores del Estado (ISSSTE), Mexico City, Mexico
| | | | - José Refugio Torres-Nava
- Servicio de Oncología, Hospital Pediátrico de Moctezuma, Secretaría de Salud del D.F., Mexico City, Mexico
| | | | - Raquel Amador-Sánchez
- Hospital General Regional No. 1 "Carlos McGregor Sánchez Navarro", IMSS, Mexico City, Mexico
| | | | - Janet Flores-Lujano
- Unidad de Investigación Médica en Epidemiología Clínica, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - María Luisa Pérez-Saldívar
- Unidad de Investigación Médica en Epidemiología Clínica, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Luis Ramiro García-López
- Servicio de Pediatría, Hospital Pediátrico de Tacubaya, Secretaría de Salud (SS), Mexico City, Mexico
| | | | | | - Haydeé Rosas-Vargas
- Unidad de Investigación Médica en Genética Humana, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", IMSS, Mexico City, Mexico
| | - Minerva Mata-Rocha
- Unidad de Investigación Médica en Genética Humana, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", IMSS, Mexico City, Mexico
| | - David Aldebarán Duarte-Rodríguez
- Unidad de Investigación Médica en Epidemiología Clínica, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Omar Alejandro Sepúlveda-Robles
- Unidad de Investigación Médica en Genética Humana, UMAE Hospital de Pediatría, Centro Médico Nacional (CMN) "Siglo XXI", IMSS, Mexico City, Mexico
| | - Ismael Mancilla-Herrera
- Departamento de infectología e inmunología, Instituto Nacional de Perinatología, Mexico City, Mexico
| | - Juan Manuel Mejía-Aranguré
- Coordinación de Investigación en Salud, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
- * E-mail: (MECM); (JMMA)
| | - Mario Ernesto Cruz-Munoz
- Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
- * E-mail: (MECM); (JMMA)
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48
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Briones JC, Espulgar WV, Koyama S, Yoshikawa H, Park J, Naito Y, Kumanogoh A, Tamiya E, Takamatsu H, Saito M. A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling. Theranostics 2020; 10:123-132. [PMID: 31903110 PMCID: PMC6929635 DOI: 10.7150/thno.37728] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Accepted: 07/24/2019] [Indexed: 12/19/2022] Open
Abstract
Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment. Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept. Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.
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49
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Lam MT, Coppola S, Krumbach OHF, Prencipe G, Insalaco A, Cifaldi C, Brigida I, Zara E, Scala S, Di Cesare S, Martinelli S, Di Rocco M, Pascarella A, Niceta M, Pantaleoni F, Ciolfi A, Netter P, Carisey AF, Diehl M, Akbarzadeh M, Conti F, Merli P, Pastore A, Levi Mortera S, Camerini S, Farina L, Buchholzer M, Pannone L, Cao TN, Coban-Akdemir ZH, Jhangiani SN, Muzny DM, Gibbs RA, Basso-Ricci L, Chiriaco M, Dvorsky R, Putignani L, Carsetti R, Janning P, Stray-Pedersen A, Erichsen HC, Horne A, Bryceson YT, Torralba-Raga L, Ramme K, Rosti V, Bracaglia C, Messia V, Palma P, Finocchi A, Locatelli F, Chinn IK, Lupski JR, Mace EM, Cancrini C, Aiuti A, Ahmadian MR, Orange JS, De Benedetti F, Tartaglia M. A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function. THE JOURNAL OF EXPERIMENTAL MEDICINE 2019. [PMID: 31601675 DOI: 10.1084/jem.20190147)] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
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Affiliation(s)
- Michael T Lam
- Department of Pediatrics, Baylor College of Medicine, Houston, TX.,Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY.,Medical Scientist Training Program and Translational Biology and Molecular Medicine Graduate Program, Baylor College of Medicine, Houston, TX
| | - Simona Coppola
- National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy
| | - Oliver H F Krumbach
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Giusi Prencipe
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Antonella Insalaco
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Cristina Cifaldi
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.,Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Immacolata Brigida
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy
| | - Erika Zara
- National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy
| | - Serena Scala
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy
| | - Silvia Di Cesare
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.,Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Simone Martinelli
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
| | - Martina Di Rocco
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.,Department of Cellular Biotechnology and Haematology, Sapienza University of Rome, Rome, Italy
| | - Antonia Pascarella
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Marcello Niceta
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Francesca Pantaleoni
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Andrea Ciolfi
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Petra Netter
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
| | - Alexandre F Carisey
- Department of Pediatrics, Baylor College of Medicine, Houston, TX.,Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY
| | - Michael Diehl
- Department of Bioengineering, Rice University, Houston, TX
| | - Mohammad Akbarzadeh
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Francesca Conti
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Pietro Merli
- Department of Pediatric Hematology and Oncology, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Anna Pastore
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Stefano Levi Mortera
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Serena Camerini
- Core Facilities, Italian National Institute of Health, Rome, Italy
| | - Luciapia Farina
- National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy.,Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Marcel Buchholzer
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Luca Pannone
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.,Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Tram N Cao
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
| | - Zeynep H Coban-Akdemir
- Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
| | - Shalini N Jhangiani
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.,Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Donna M Muzny
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.,Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Richard A Gibbs
- Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.,Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Luca Basso-Ricci
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy
| | - Maria Chiriaco
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Radovan Dvorsky
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Lorenza Putignani
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Rita Carsetti
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Petra Janning
- Department of Chemical Biology, Max-Planck Institute of Molecular Physiology, Dortmund, Germany
| | - Asbjorg Stray-Pedersen
- Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.,Norwegian National Unit for Newborn Screening, Department of Pediatric and Adolescent Medicine, Oslo University Hospital, Oslo, Norway.,Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | | | - AnnaCarin Horne
- Pediatric Rheumatology, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, Sweden.,Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
| | - Yenan T Bryceson
- Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.,Broegelmann Research Laboratory, Institute of Clinical Sciences, University of Bergen, Bergen, Norway
| | - Lamberto Torralba-Raga
- Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
| | - Kim Ramme
- Pediatric Hematology, Immunology and HCT Section, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, Sweden
| | - Vittorio Rosti
- Center for Myelofibrosis, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy
| | - Claudia Bracaglia
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Virginia Messia
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Paolo Palma
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Andrea Finocchi
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.,Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Franco Locatelli
- Department of Pediatric Hematology and Oncology, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.,Department of Pediatrics, Sapienza University of Rome, Italy
| | - Ivan K Chinn
- Department of Pediatrics, Baylor College of Medicine, Houston, TX.,Division of Pediatric Immunology, Allergy, Rheumatology, Department of Pediatrics, Baylor College of Medicine, Houston, TX
| | - James R Lupski
- Department of Pediatrics, Baylor College of Medicine, Houston, TX.,Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.,Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Emily M Mace
- Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY
| | - Caterina Cancrini
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.,Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Alessandro Aiuti
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy.,Pediatric Immunohematology, San Raffaele Scientific Institute, Milan, Italy.,Vita Salute San Raffaele University, Milan, Italy
| | - Mohammad R Ahmadian
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Jordan S Orange
- Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY .,Medical Scientist Training Program and Translational Biology and Molecular Medicine Graduate Program, Baylor College of Medicine, Houston, TX
| | - Fabrizio De Benedetti
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Marco Tartaglia
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
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Lam MT, Coppola S, Krumbach OHF, Prencipe G, Insalaco A, Cifaldi C, Brigida I, Zara E, Scala S, Di Cesare S, Martinelli S, Di Rocco M, Pascarella A, Niceta M, Pantaleoni F, Ciolfi A, Netter P, Carisey AF, Diehl M, Akbarzadeh M, Conti F, Merli P, Pastore A, Levi Mortera S, Camerini S, Farina L, Buchholzer M, Pannone L, Cao TN, Coban-Akdemir ZH, Jhangiani SN, Muzny DM, Gibbs RA, Basso-Ricci L, Chiriaco M, Dvorsky R, Putignani L, Carsetti R, Janning P, Stray-Pedersen A, Erichsen HC, Horne A, Bryceson YT, Torralba-Raga L, Ramme K, Rosti V, Bracaglia C, Messia V, Palma P, Finocchi A, Locatelli F, Chinn IK, Lupski JR, Mace EM, Cancrini C, Aiuti A, Ahmadian MR, Orange JS, De Benedetti F, Tartaglia M. A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function. J Exp Med 2019; 216:2778-2799. [PMID: 31601675 PMCID: PMC6888978 DOI: 10.1084/jem.20190147] [Citation(s) in RCA: 146] [Impact Index Per Article: 24.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Revised: 04/19/2019] [Accepted: 08/06/2019] [Indexed: 12/27/2022] Open
Abstract
Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
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Affiliation(s)
- Michael T Lam
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
- Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY
- Medical Scientist Training Program and Translational Biology and Molecular Medicine Graduate Program, Baylor College of Medicine, Houston, TX
| | - Simona Coppola
- National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy
| | - Oliver H F Krumbach
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Giusi Prencipe
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Antonella Insalaco
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Cristina Cifaldi
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
- Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Immacolata Brigida
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy
| | - Erika Zara
- National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy
| | - Serena Scala
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy
| | - Silvia Di Cesare
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
- Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Simone Martinelli
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
| | - Martina Di Rocco
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
- Department of Cellular Biotechnology and Haematology, Sapienza University of Rome, Rome, Italy
| | - Antonia Pascarella
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Marcello Niceta
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Francesca Pantaleoni
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Andrea Ciolfi
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Petra Netter
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
| | - Alexandre F Carisey
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
- Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY
| | - Michael Diehl
- Department of Bioengineering, Rice University, Houston, TX
| | - Mohammad Akbarzadeh
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Francesca Conti
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Pietro Merli
- Department of Pediatric Hematology and Oncology, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Anna Pastore
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Stefano Levi Mortera
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Serena Camerini
- Core Facilities, Italian National Institute of Health, Rome, Italy
| | - Luciapia Farina
- National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Marcel Buchholzer
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Luca Pannone
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Tram N Cao
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
| | - Zeynep H Coban-Akdemir
- Baylor-Hopkins Center for Mendelian Genomics, Houston, TX
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
| | - Shalini N Jhangiani
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
- Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Donna M Muzny
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
- Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Richard A Gibbs
- Baylor-Hopkins Center for Mendelian Genomics, Houston, TX
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
- Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Luca Basso-Ricci
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy
| | - Maria Chiriaco
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Radovan Dvorsky
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Lorenza Putignani
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Rita Carsetti
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Petra Janning
- Department of Chemical Biology, Max-Planck Institute of Molecular Physiology, Dortmund, Germany
| | - Asbjorg Stray-Pedersen
- Baylor-Hopkins Center for Mendelian Genomics, Houston, TX
- Norwegian National Unit for Newborn Screening, Department of Pediatric and Adolescent Medicine, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | | | - AnnaCarin Horne
- Pediatric Rheumatology, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, Sweden
- Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
| | - Yenan T Bryceson
- Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
- Broegelmann Research Laboratory, Institute of Clinical Sciences, University of Bergen, Bergen, Norway
| | - Lamberto Torralba-Raga
- Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
| | - Kim Ramme
- Pediatric Hematology, Immunology and HCT Section, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, Sweden
| | - Vittorio Rosti
- Center for Myelofibrosis, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy
| | - Claudia Bracaglia
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Virginia Messia
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Paolo Palma
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Andrea Finocchi
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
- Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Franco Locatelli
- Department of Pediatric Hematology and Oncology, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
- Department of Pediatrics, Sapienza University of Rome, Italy
| | - Ivan K Chinn
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
- Division of Pediatric Immunology, Allergy, Rheumatology, Department of Pediatrics, Baylor College of Medicine, Houston, TX
| | - James R Lupski
- Department of Pediatrics, Baylor College of Medicine, Houston, TX
- Baylor-Hopkins Center for Mendelian Genomics, Houston, TX
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
- Human Genome Sequencing Center of Baylor College of Medicine, Houston, TX
| | - Emily M Mace
- Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY
| | - Caterina Cancrini
- Academic Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
- Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Alessandro Aiuti
- San Raffaele Telethon Institute for Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Scientific Institute, Milan, Italy
- Pediatric Immunohematology, San Raffaele Scientific Institute, Milan, Italy
- Vita Salute San Raffaele University, Milan, Italy
| | - Mohammad R Ahmadian
- Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - Jordan S Orange
- Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY
- Medical Scientist Training Program and Translational Biology and Molecular Medicine Graduate Program, Baylor College of Medicine, Houston, TX
| | - Fabrizio De Benedetti
- Immunology Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Marco Tartaglia
- Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
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