Characterization of human lymphoblastoid cell lines as a novel in vitro test system to predict the immunotoxicity of xenobiotics

Markovič, Tijana; Gobec, Martina; Genevieve, David; Mlinarič-Raščan, Irena

doi:10.1016/j.toxlet.2014.12.013

Cited by 15 publications

(5 citation statements)

References 28 publications

(52 reference statements)

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“…Activation of T lymphocytes has also been analyzed based on cytokine secretion in supernatant from PBMCs or direct from the cells in the whole blood by using ELISA ( 25 , 26 ). Also human lymphoblastoid cell line was used to assess cytokine production for assessment of four immunotoxic compounds; tributyltin chloride, cyclosporine A, benzo(a)pyrene and verapamil hydrochloride ( 27 ). This however was performed by ELISA and the cells were unspecifically stimulated with PMA/ionomycin.…”

Section: Discussionmentioning

confidence: 99%

A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

Pierzchalski,

Zenclussen,

Herberth

2024

Front. Immunol.

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BackgroundThere is a growing need for immunological assays to test toxic and modulatory effects of chemicals. The assays should be easy to use, reproducible and superior to cell line-based assays. We have therefore developed a comprehensive portfolio of assays based on primary human blood cells that are suitable for testing chemical effects.MethodsThe flow cytometry-based assays were designed to target a wide range of human peripheral blood mononuclear cells and whole blood, including T cells, NK cells, B cells, basophils and innate-like T cells such as γδT, MAIT and NKT cells. We have selected a set of activation markers for each immune cell, e.g: CD154 (T cells), CD137, CD107a (NK cells), CD63 (basophils), CD69, CD83 (B cells), CD69, IFN-γ (MAIT cells) and we selected cell specific stimuli: aCD3 antibodies (T cells); E. coli and cytokines IL-12/15/18 (MAIT cells); CpG ODN2006, R848 or aCD40 antibodies (B cells), fMLP or aFcϵR1 (basophils) or K562 cells (NK cells).ResultsBy selecting immune cell-specific markers and cell-specific stimuli, we were able to induce particular immune responses from the targeted immune cells. For example, the response to stimulation with anti-CD3 antibodies was in 36.8% of CD107a+CD8+ cells. Cytokine stimulation induced the production of IFN-γ in 30% of MAIT cells. After stimulation with E. coli, around 50% of MAIT cells produced TNF. About 40% of basophils responded to aFcƐR1 stimulation. Similar activation ranges were achieved in K562-stimulated NK cells.ConclusionOur test portfolio covers the most relevant immune cells present in human blood, providing a solid basis for in vitro toxicity and immunomodulatory testing of chemicals. By using human blood, the natural composition of cells found in the blood can be determined and the effects of chemicals can be detected at the cellular level.

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Section: Discussionmentioning

confidence: 99%

A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

Pierzchalski,

Zenclussen,

Herberth

2024

Front. Immunol.

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“…A more likely explanation relates to the metabolic capacity of the 3A9 and Ch27 cells. Both CYPH and BAP are known to require biotransformation by cytochrome P450 isozymes (Connors et al, 1974, Dean et al, 1983, Carfi et al, 2007, Markovic et al, 2015. However, the mechanism of AZPR biotransformation involves conjugation with glutathione rather than cytochrome P450 activity (Maltzman et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…The effect of known immunosuppressive chemicals with different mechanisms of action on IL-2 production was investigated (Table 1). Treatment with CYA, a well-known immunosuppressive drug (Carfi et al, 2007, Markovic et al, 2015, completely eliminated detectable IL-2 release from 3A9 T cells co-cultured with antigen-bearing Ch27 B cells ( Figure 5A) with an IC 25 and IC 50 for IL-2 production of 1.19 nM and 1.99 nM, respectively (Table 2). Treatment with other immunosuppressant compounds (i.e.…”

Section: Immunosuppressive Chemicals With Different Mechanisms Of Actmentioning

confidence: 99%

Development and utilization of a unique in vitro antigen presentation co-culture model for detection of immunomodulating substances

Lehmann

Williams

2018

Toxicology in Vitro

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Current regulatory immunotoxicity studies require the use of animal models. However, evolving regulatory requirements, the need to evaluate large numbers of chemicals efficiently and societal pressures are driving the development and utilization of alternative in vitro methods for identifying potential immunotoxicants. In line with these efforts, we developed a novel in vitro cell-based assay to evaluate effects on antigen presentation - a key step in successful immunization. In this assay, Ch27 B cells acquire and present hen egg lysozyme peptides to antigen-restricted 3A9 T cells, causing them to produce and secrete IL-2. IL-2 levels in the culture medium may be monitored to identify effects of immunotoxicant exposure on antigen uptake, processing or presentation by the Ch27 cells and on antigen recognition and IL-2 production and secretion by the 3A9 cells. IL-2 production was reduced in response to treatment with well-known immunotoxicants cyclosporin A (CYA), dexamethasone (DEX), azathioprine (AZPR), methotrexate (MOT) and benzo(a)pyrene (BAP) but was not affected by treatment with cyclophosphamide (CYPH). A negative control compound mannitol (MANN) altered neither cell viability nor IL-2 levels whereas the lysosomotrophic compound ammonium chloride (AMCL) reduced IL-2 production. This novel in vitro assay of immune function may be suitable for integration into a tiered testing battery for screening and prioritization of potential immunosuppressants.

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“…A panel of cytokines that change independently of the immunosuppressive mechanism will be selected as markers for immunosuppression. In the more advanced stages of the project, the lymphoblastoid cell line LCL ( Markovič et al, 2015 ) will be added to the co-culture to bring the model closer to whole blood. Once the model is in place, we will test whether endocrine disruptors such as bisphenols and PFAS act as immunosuppressants.…”

Section: Immunosuppressionmentioning

confidence: 99%

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals — a PARC (Partnership for the Assessment of Risk from Chemicals) project

Snapkow,

Smith,

Arnesdotter

et al. 2024

Front. Toxicol.

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As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

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Characterization of human lymphoblastoid cell lines as a novel in vitro test system to predict the immunotoxicity of xenobiotics

Cited by 15 publications

References 28 publications

A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

Development and utilization of a unique in vitro antigen presentation co-culture model for detection of immunomodulating substances

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals — a PARC (Partnership for the Assessment of Risk from Chemicals) project

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