Methods in cell biology最新文献_第2页

In vitro ILC differentiation from human HSCs. 人造血干细胞的体外ILC分化。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-19 DOI: 10.1016/bs.mcb.2024.10.004

Silvia Santopolo, Cecilia Ciancaglini, Francesca Romana Mariotti, Lorenzo Moretta, Linda Quatrini

{"title":"In vitro ILC differentiation from human HSCs.","authors":"Silvia Santopolo, Cecilia Ciancaglini, Francesca Romana Mariotti, Lorenzo Moretta, Linda Quatrini","doi":"10.1016/bs.mcb.2024.10.004","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.004","url":null,"abstract":"The Innate Lymphoid Cells (ILCs) are a family of innate immune cells composed by the Natural Killer (NK) cells and the helper ILCs (hILCs) (ILC1, ILC2, ILC3), both developing from a common ILC precursor (ILCP) derived from hematopoietic stem cells (HSCs). A correct ILC reconstitution is crucial, particularly in patients receiving HSC transplantation (HSCT), the only therapeutic option for many adult and pediatric high-risk hematological malignancies. Indeed, mainly thanks to their cytotoxic activity, NK cells have a strong Graft-versus-Leukemia (GvL) effect. On the other hand, hILCs, that are mainly tissue resident, are involved in tissue repair and homeostasis, Graft-versus-Host Disease (GvHD) prevention and immune response to infections. Unlike NK cell development, hILC-poiesis is still poorly characterized in humans. Here, we provide a protocol for the in vitro ILC differentiation from healthy donor peripheral blood-derived CD34+ HSCs. This could represent a useful model to dissect the molecular mechanisms by which the distinct ILC subsets are generated from ILCP leading to the development of novel strategies to improve the HSCT clinical outcome.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"41-57"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008130","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Retrovirus-based manufacturing of chimeric antigen receptor-modified T cells for cancer therapy research. 基于逆转录病毒制造嵌合抗原受体修饰的T细胞用于癌症治疗研究。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-19 DOI: 10.1016/bs.mcb.2024.10.017

Sophia Stock, Luisa Fertig, Vivien Doreen Menkhoff, Thaddäus Strzalkowski, Manuel Caruso, Sebastian Kobold

{"title":"Retrovirus-based manufacturing of chimeric antigen receptor-modified T cells for cancer therapy research.","authors":"Sophia Stock, Luisa Fertig, Vivien Doreen Menkhoff, Thaddäus Strzalkowski, Manuel Caruso, Sebastian Kobold","doi":"10.1016/bs.mcb.2024.10.017","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.017","url":null,"abstract":"Treatment with autologous chimeric antigen receptor (CAR)-modified T cells can achieve outstanding clinical response rates in heavily pretreated patients with B and plasma cell malignancies. However, relapses occur, and they limit the efficacy of this promising treatment approach. The complex GMP-compliant production and high treatment costs cause that CAR T cells cannot yet be used in a broad population. Among others, CAR T cell therapy has evolved regarding vector design and manufacturing process. Optimal production of CAR T cells is not yet defined, far from being standardized. Quality, cellular composition and immunophenotype of the administered CAR T cells are influenced by the manufacturing protocol and therefore play a crucial role for therapeutic success. For the gene transfer, viral and non-viral strategies are available. Retrovirus-based protocols for CAR T cell production offer advantages in terms of stable gene integration, sufficient transduction efficiency, proven clinical success, and scalability. Here, we detail a retrovirus-based generation protocol of human CAR-modified T cells for experimental immunotherapeutic treatment of cancer cells. For the CAR generation, HEK-293-based packaging cell lines, CD3+ selection, CD3/CD28-coated bead-based activation and IL-2/IL-15-mediated expansion were used. This protocol can be applied for every possible CAR construct after being successfully transfected in HEK-293-based packaging cell lines.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"329-352"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Stereotactic injection of murine brain tumor cells for neuro-oncology studies.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-08-12 DOI: 10.1016/bs.mcb.2024.07.005

Camille Daviaud, María Cecilia Lira, Claire Vanpouille-Box, Mara De Martino

引用次数: 0

Measuring interaction force between T lymphocytes and their target cells using live microscopy and laminar shear flow chambers.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-08 DOI: 10.1016/bs.mcb.2024.09.001

Sophie Goyard, Amandine Schneider, Jerko Ljubetic, Nicolas Inacio, Marie Juzans, Céline Cuche, Pascal Bochet, Vincenzo Di Bartolo, Andrés Alcover, Thierry Rose

{"title":"Measuring interaction force between T lymphocytes and their target cells using live microscopy and laminar shear flow chambers.","authors":"Sophie Goyard, Amandine Schneider, Jerko Ljubetic, Nicolas Inacio, Marie Juzans, Céline Cuche, Pascal Bochet, Vincenzo Di Bartolo, Andrés Alcover, Thierry Rose","doi":"10.1016/bs.mcb.2024.09.001","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.09.001","url":null,"abstract":"Understanding the immunological synapse formation and dynamics can be enriched by measuring cell-cell interaction forces and their kinetics. Microscopy imaging reveals structural organization of the synapse, while physical methods detail its mechanical construction. Various techniques have been reported for measuring forces needed to rupture the interface between a T lymphocyte and its target cell but most of them measure one pair at a time. We describe here a laminar shear flow-based method that exerts dragging forces on T cell-target cells pairs immobilized on the surface of a flow chamber. Increasing flow rate allows us to observe the detachment of hundreds of cell conjugates on the wide field of a light transmission microscope. Monitoring precisely the flow rate gradient exerted on T cells readily yields synapse rupture measurements. Dragging forces are measured at the point of rupture as a linear function of the flow speed in minutes from 10pN to 20nN for each cell pair among a statistically representative cell population in the whole field of view of a single experiment. The output cells can be collected in multi-well plate sorted in the increasing order of rupture forces. We used this approach to unveil the involvement of the cytoskeleton regulator adenomatous polyposis coli (APC) in the stability of immunological synapses formed between human cytotoxic T cell and tumor target cells. APC is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Reduced APC expression impairs T cell adhesion with tumor target cells suggesting an impact of APC mutation in anti-tumor immune defense.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"175-200"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Antimicrobial regime for gut microbiota depletion in experimental mice models.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-07-01 DOI: 10.1016/bs.mcb.2024.05.005

Laura Carrillo-Serradell, Alessandra Borgognone, Marc Noguera-Julian, Violeta Planells-Romeo, Lucía Aragón-Serrano, Mariona Parera, Francesc Català-Moll, Sergi Casadó-Llombart, María Velasco-de Andrés, Roger Paredes, Francisco Lozano

{"title":"Antimicrobial regime for gut microbiota depletion in experimental mice models.","authors":"Laura Carrillo-Serradell, Alessandra Borgognone, Marc Noguera-Julian, Violeta Planells-Romeo, Lucía Aragón-Serrano, Mariona Parera, Francesc Català-Moll, Sergi Casadó-Llombart, María Velasco-de Andrés, Roger Paredes, Francisco Lozano","doi":"10.1016/bs.mcb.2024.05.005","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.05.005","url":null,"abstract":"Mice models serve as a valuable tool to study microbiome-immune system interactions. While the use of germ-free mice may represent the gold-standard method, antibiotic-based microbiome depletion provides a more cost-efficient and feasible system. The protocol here in presented provides a mild antimicrobial regime to deplete basal microbiota in 8-week-old C57BL/6 mice, aiming to ensure reproducibility in microbiota studies. The antibiotic regime combines five different antimicrobial agents delivered either ad libitum or via oral gavage, aiming at depleting core gut microbiota in mice. Various administration timings were explored, concluding there were no differences when the antimicrobial treatment was applied for 3, 5 or 7 consecutive days. By offering a detailed antimicrobial preparation and mouse administration, as well as fecal sample processing and 16s rRNA sequencing, this protocol provides an initial framework to develop mice microbiota studies, with perceptible results in fecal microbiota composition.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"101-114"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Characterizing tumor-infiltrating group 1 innate lymphoid cells in PyMT breast tumors.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-04-24 DOI: 10.1016/bs.mcb.2024.03.008

Douglas C Chung, Alisha R Elford, Nicolas Jacquelot

引用次数: 0

Evaluating polyglutamine protein aggregation and toxicity in transgenic Caenorhabditis elegans models of Huntington's disease.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-07-09 DOI: 10.1016/bs.mcb.2024.06.002

Larissa Marafiga Cordeiro, Félix Alexandre Antunes Soares, Leticia Priscilla Arantes

{"title":"Evaluating polyglutamine protein aggregation and toxicity in transgenic Caenorhabditis elegans models of Huntington's disease.","authors":"Larissa Marafiga Cordeiro, Félix Alexandre Antunes Soares, Leticia Priscilla Arantes","doi":"10.1016/bs.mcb.2024.06.002","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.06.002","url":null,"abstract":"Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by a repeat of the cytosine-adenine-guanine trinucleotide (CAG) in the huntingtin gene (HTT). This results in the translation of a mutant huntingtin (mHTT) protein with an abnormally long polyglutamine (polyQ) repeat. The pathology of HD leads to neuronal cell loss, motor abnormalities, and dementia. Currently, the pathogenesis of HD remains incompletely understood, and available treatments only address symptoms. Caenorhabditis elegans has been used as a model for neurodegenerative diseases, enabling the exploration of the molecular, cellular, and physiological mechanisms underlying HD pathogenesis. It also facilitates the investigation of potential therapeutic targets and interventions. Here, we describe common experiments employed to assess polyQ aggregation and toxicity in transgenic C. elegans models of HD, utilizing fluorescent markers to detect protein aggregation and neuron degeneration, in addition to specific behavioral assays (thrash frequency, nose touch response, and octanol response).","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"115-130"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Matching model with mechanism: Appropriate rodent models for studying various aspects of diabetes pathophysiology.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-06-19 DOI: 10.1016/bs.mcb.2024.05.003

Lydia F Daniels Gatward, Aileen J F King

{"title":"Matching model with mechanism: Appropriate rodent models for studying various aspects of diabetes pathophysiology.","authors":"Lydia F Daniels Gatward, Aileen J F King","doi":"10.1016/bs.mcb.2024.05.003","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.05.003","url":null,"abstract":"Many rodent models are available for preclinical diabetes research making it a challenge for researchers to choose the most appropriate one for their experimental question. To aid in this, models have classically been categorized according to which type of diabetes they represent, and further into whether the model is induced, spontaneous or the result of genetic manipulation. This fails to capture the complexity of pathogenesis seen in diabetes in humans. This includes pathogenesis specifically involving the beta cell, which is no longer considered to be innocuous in the development and progression of diabetes. In this chapter we explore rodent models that incorporate the initiating factors believed to be involved in type 1 diabetes (autoimmunity) and type 2 diabetes (insulin resistance), before further discussing rodents that can be used to model specific mechanisms involved in a failure of functional beta cell mass (impaired beta cell function and beta cell apoptosis). We segregate models of beta cell pathogenesis based on the beta cell stressor predominantly associated with phenotype, but it is important to consider that most rodent models will exhibit more than one beta cell stressor. Similarly, many models exhibit more than one pathogenic mechanism, for example the same model may show insulin resistance, impaired beta cell function as well as beta cell loss. This can complicate interpretation of results and should be considered, and the model thoroughly researched, during the experimental planning stage.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"39-68"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation of human and murine exhausted CD8⁺ T cells in vitro. 体外培养人和小鼠耗竭CD8+ T细胞。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-13 DOI: 10.1016/bs.mcb.2024.10.007

Rosa Ana Lacalle, Raquel Blanco, Rebeca García-Lucena, Santos Mañes

{"title":"Generation of human and murine exhausted CD8+ T cells in vitro.","authors":"Rosa Ana Lacalle, Raquel Blanco, Rebeca García-Lucena, Santos Mañes","doi":"10.1016/bs.mcb.2024.10.007","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.007","url":null,"abstract":"T cell exhaustion is a state of dysfunction that can occur due to persistent exposure to antigens, such as in the tumor microenvironment. The progressive loss of effector functions in exhausted T cells can lead to resistance to immune checkpoint inhibitors and adoptive cell immunotherapies. Improving our understanding of the exhaustion process is thus crucial for optimizing the clinical outcomes of immunotherapy. A significant hurdle in this area is obtaining an adequate quantity of exhausted T cells. One solution could be the in vitro production of exhausted T cells by mimicking exhaustion-induced conditions. We present a simple, repeatable, flow cytometry-assisted method for generating exhausted CD8+ T cells from both human and mouse sources. This flexible protocol works with various cell sources and activation methods. Our results confirm the production of dysfunctional CD8+ T cells, akin to those in mouse tumor models and patient tumor samples. This methodology could help identify genes involved in the exhaustion process and serve as a platform for finding agents capable of altering, reversing, or accelerating this dysfunctional state. By using both mouse and human models, we increase the adaptability of the method, making it a powerful instrument for assessing potential substances with immunotherapeutic utility.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"93-114"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation. 解读人类血液和肿瘤中中性粒细胞的异质性：分离中性粒细胞和评估其对t细胞增殖影响的方法。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-25 DOI: 10.1016/bs.mcb.2024.10.010

Nicolas Delhez, Frank Aboubakar Nana, Camille Houbion, Alexandre Bayard, Annika Bruger, Christophe Vanhaver, Sven Brandau, Pierre van der Bruggen, Thibault Hirsch

{"title":"Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation.","authors":"Nicolas Delhez, Frank Aboubakar Nana, Camille Houbion, Alexandre Bayard, Annika Bruger, Christophe Vanhaver, Sven Brandau, Pierre van der Bruggen, Thibault Hirsch","doi":"10.1016/bs.mcb.2024.10.010","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.010","url":null,"abstract":"Neutrophils were historically considered a homogenous population of cells with functions limited to innate immunity against external threats. However, with the rise of immunotherapy, recent works have shown that neutrophils are also important actors in immuno-oncology. In this context, neutrophils appear as a more heterogenous population of cells. However, many reported neutrophil subpopulations, or neutrophils with various transcriptional states, lack functional characterization to confirm their suspected roles. Thus, we believe that functional assays remain essential to define the role of neutrophils in cancer. In this chapter, we present a T-cell proliferation assay based on the use of allogeneic T-cells to assess the suppressive capabilities of neutrophils isolated from human blood or tumor samples. Allogeneic T-cells are isolated in large quantities from the blood of non-cancerous donors and frozen in aliquots to be used in several experiments. This reduces variability by excluding other cancer-derived factors, which would be present if autologous T-cell were used and allows to isolate the effect of neutrophils on T-cell proliferation. Thawed T-cells have poor proliferative capacities and to initiate proliferation they require co-culture with mature dendritic cells that we generate from monocytes isolated from the same blood sample. Initially developed for lung cancer patients, our method to isolate low-density neutrophils (LDN) and normal-density neutrophils (NDN) can be used with any patient and adapted to other kind of samples (e.g., ascites, urine, …).","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"151-196"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0