Marta Valente Pinto , Alex-Mikael Barkoff , Sagida Bibi , Aapo Knuutila , Johanna Teräsjärvi , Elizabeth Clutterbuck , Sophie Gimenez-Fourage , Anke Pagnon , Jacqueline A.M. van Gaans-van den Brink , Veronique Corbiere , Aymeric De Montfort , Anja Saso , Haddijatou Jobe , Sophie Roetynck , Beate Kampmann , Elles Simonetti , Dimitri Diavatopoulos , Eleonora E. Lambert , Jussi Mertsola , Pascal Blanc , Ronald de Groot
{"title":"一种新型全血测定法，用于量化百日咳博德特氏菌抗原反应中 T 细胞相关细胞因子的释放。","authors":"Marta Valente Pinto , Alex-Mikael Barkoff , Sagida Bibi , Aapo Knuutila , Johanna Teräsjärvi , Elizabeth Clutterbuck , Sophie Gimenez-Fourage , Anke Pagnon , Jacqueline A.M. van Gaans-van den Brink , Veronique Corbiere , Aymeric De Montfort , Anja Saso , Haddijatou Jobe , Sophie Roetynck , Beate Kampmann , Elles Simonetti , Dimitri Diavatopoulos , Eleonora E. Lambert , Jussi Mertsola , Pascal Blanc , Ronald de Groot","doi":"10.1016/j.jim.2024.113758","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><div><em>Bordetella pertussis</em> continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination.</div></div><div><h3>Material and methods</h3><div>Longitudinal WB samples were collected from a small set of subjects (<em>n</em> = 38) aged 7–70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated <em>B. pertussis</em> lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay.</div></div><div><h3>Results</h3><div>The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles.</div></div><div><h3>Conclusions</h3><div>The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6000,"publicationDate":"2024-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens\",\"authors\":\"Marta Valente Pinto , Alex-Mikael Barkoff , Sagida Bibi , Aapo Knuutila , Johanna Teräsjärvi , Elizabeth Clutterbuck , Sophie Gimenez-Fourage , Anke Pagnon , Jacqueline A.M. van Gaans-van den Brink , Veronique Corbiere , Aymeric De Montfort , Anja Saso , Haddijatou Jobe , Sophie Roetynck , Beate Kampmann , Elles Simonetti , Dimitri Diavatopoulos , Eleonora E. Lambert , Jussi Mertsola , Pascal Blanc , Ronald de Groot\",\"doi\":\"10.1016/j.jim.2024.113758\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><div><em>Bordetella pertussis</em> continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination.</div></div><div><h3>Material and methods</h3><div>Longitudinal WB samples were collected from a small set of subjects (<em>n</em> = 38) aged 7–70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated <em>B. pertussis</em> lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay.</div></div><div><h3>Results</h3><div>The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles.</div></div><div><h3>Conclusions</h3><div>The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. 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A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens
Background
Bordetella pertussis continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination.
Material and methods
Longitudinal WB samples were collected from a small set of subjects (n = 38) aged 7–70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated B. pertussis lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay.
Results
The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles.
Conclusions
The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future.
期刊介绍：
The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells.
In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.
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