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A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses
INTRODUCTION: The characterization of B. pertussis (Bp) antigen-specific CD4(+) T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays. METHODS: To overcome this limitation, we developed a...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9939445/ https://www.ncbi.nlm.nih.gov/pubmed/36814927 http://dx.doi.org/10.3389/fimmu.2023.1101366 |
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author | Corbière, Véronique Lambert, Eleonora E. Rodesch, Marine van Gaans-van den Brink, Jacqueline A. M. Misiak, Alicja Simonetti, Elles Van Praet, Anne Godefroid, Audrey Diavatopoulos, Dimitri A. van Els, Cécile A. C. M. Mascart, Françoise |
author_facet | Corbière, Véronique Lambert, Eleonora E. Rodesch, Marine van Gaans-van den Brink, Jacqueline A. M. Misiak, Alicja Simonetti, Elles Van Praet, Anne Godefroid, Audrey Diavatopoulos, Dimitri A. van Els, Cécile A. C. M. Mascart, Françoise |
author_sort | Corbière, Véronique |
collection | PubMed |
description | INTRODUCTION: The characterization of B. pertussis (Bp) antigen-specific CD4(+) T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays. METHODS: To overcome this limitation, we developed a two-step assay comprising a short-term stimulation of fresh whole blood with Bp antigens and cryopreservation of the stimulated cells, followed later on by batch-wise intracellular cytokine analysis by flow cytometry. Blood samples collected from recently acellular (aP) vaccine boosted subjects with a whole-cell- or aP-primed background was incubated for 24 hrs with Pertussis toxin, Filamentous hemagglutinin or a Bp lysate (400µl per stimulation). Antigen-specific IFN-γ-, IL-4/IL-5/IL-13-, IL-17A/IL-17F- and/or IL-22-producing CD4(+) T cells were quantified by flow cytometry to reveal Th1, Th2, and Th17-type responses, respectively. The frequencies of IFN-γ-producing CD8(+) T cells were also analyzed. RESULTS: We demonstrate high reproducibility of the Bp-specific whole blood intracellular staining assay. The results obtained after cryopreservation of the stimulated and fixed cells were very well correlated to those obtained without cryopreservation, an approach used in our previously published assay. Optimization resulted in high sensitivity thanks to very low non-specific backgrounds, with reliable detection of Bp antigen-specific Th1, Th2 and Th17-type CD4(+) T cells, in the lowest range frequency of 0.01-0.03%. Bp antigen-specific IFN-γ(+) CD8(+) T lymphocytes were also detected. This test is easy to perform, analyse and interpret with the establishment of strict criteria defining Bp antigen responses. DISCUSSION: Thus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4(+) T cells induced by current and next generation pertussis vaccines. |
format | Online Article Text |
id | pubmed-9939445 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-99394452023-02-21 A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses Corbière, Véronique Lambert, Eleonora E. Rodesch, Marine van Gaans-van den Brink, Jacqueline A. M. Misiak, Alicja Simonetti, Elles Van Praet, Anne Godefroid, Audrey Diavatopoulos, Dimitri A. van Els, Cécile A. C. M. Mascart, Françoise Front Immunol Immunology INTRODUCTION: The characterization of B. pertussis (Bp) antigen-specific CD4(+) T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays. METHODS: To overcome this limitation, we developed a two-step assay comprising a short-term stimulation of fresh whole blood with Bp antigens and cryopreservation of the stimulated cells, followed later on by batch-wise intracellular cytokine analysis by flow cytometry. Blood samples collected from recently acellular (aP) vaccine boosted subjects with a whole-cell- or aP-primed background was incubated for 24 hrs with Pertussis toxin, Filamentous hemagglutinin or a Bp lysate (400µl per stimulation). Antigen-specific IFN-γ-, IL-4/IL-5/IL-13-, IL-17A/IL-17F- and/or IL-22-producing CD4(+) T cells were quantified by flow cytometry to reveal Th1, Th2, and Th17-type responses, respectively. The frequencies of IFN-γ-producing CD8(+) T cells were also analyzed. RESULTS: We demonstrate high reproducibility of the Bp-specific whole blood intracellular staining assay. The results obtained after cryopreservation of the stimulated and fixed cells were very well correlated to those obtained without cryopreservation, an approach used in our previously published assay. Optimization resulted in high sensitivity thanks to very low non-specific backgrounds, with reliable detection of Bp antigen-specific Th1, Th2 and Th17-type CD4(+) T cells, in the lowest range frequency of 0.01-0.03%. Bp antigen-specific IFN-γ(+) CD8(+) T lymphocytes were also detected. This test is easy to perform, analyse and interpret with the establishment of strict criteria defining Bp antigen responses. DISCUSSION: Thus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4(+) T cells induced by current and next generation pertussis vaccines. Frontiers Media S.A. 2023-02-06 /pmc/articles/PMC9939445/ /pubmed/36814927 http://dx.doi.org/10.3389/fimmu.2023.1101366 Text en Copyright © 2023 Corbière, Lambert, Rodesch, van Gaans-van den Brink, Misiak, Simonetti, Van Praet, Godefroid, Diavatopoulos, van Els, Mascart and PERISCOPE WP5 Task 7 working group https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Immunology Corbière, Véronique Lambert, Eleonora E. Rodesch, Marine van Gaans-van den Brink, Jacqueline A. M. Misiak, Alicja Simonetti, Elles Van Praet, Anne Godefroid, Audrey Diavatopoulos, Dimitri A. van Els, Cécile A. C. M. Mascart, Françoise A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses |
title | A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses |
title_full | A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses |
title_fullStr | A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses |
title_full_unstemmed | A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses |
title_short | A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses |
title_sort | semi high-throughput whole blood-based flow cytometry assay to detect and monitor bordetella pertussis-specific th1, th2 and th17 responses |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9939445/ https://www.ncbi.nlm.nih.gov/pubmed/36814927 http://dx.doi.org/10.3389/fimmu.2023.1101366 |
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