A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus.
<h4>Background</h4>Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2023-03-01
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| Series: | PLoS Neglected Tropical Diseases |
| Online Access: | https://doi.org/10.1371/journal.pntd.0010905 |
| _version_ | 1827968481110261760 |
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| author | Manutsanun Inthawong Nattapon Pinthong Areerat Thaiprakhong Tri Wangrangsimakul Piyanate Sunyakumthorn Jennifer Hill Piengchan Sonthayanon Daniel H Paris Susanna J Dunachie Barbara Kronsteiner |
| author_facet | Manutsanun Inthawong Nattapon Pinthong Areerat Thaiprakhong Tri Wangrangsimakul Piyanate Sunyakumthorn Jennifer Hill Piengchan Sonthayanon Daniel H Paris Susanna J Dunachie Barbara Kronsteiner |
| author_sort | Manutsanun Inthawong |
| collection | DOAJ |
| description | <h4>Background</h4>Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources.<h4>Methodology/principle findings</h4>We optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients.<h4>Conclusions/significance</h4>In conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level. |
| first_indexed | 2024-04-09T18:22:17Z |
| format | Article |
| id | doaj.art-87f458c6a3c342ad862f6712215d3c35 |
| institution | Directory Open Access Journal |
| issn | 1935-2727 1935-2735 |
| language | English |
| last_indexed | 2024-04-09T18:22:17Z |
| publishDate | 2023-03-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Neglected Tropical Diseases |
| spelling | doaj.art-87f458c6a3c342ad862f6712215d3c352023-04-12T05:33:31ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352023-03-01173e001090510.1371/journal.pntd.0010905A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus.Manutsanun InthawongNattapon PinthongAreerat ThaiprakhongTri WangrangsimakulPiyanate SunyakumthornJennifer HillPiengchan SonthayanonDaniel H ParisSusanna J DunachieBarbara Kronsteiner<h4>Background</h4>Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources.<h4>Methodology/principle findings</h4>We optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients.<h4>Conclusions/significance</h4>In conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level.https://doi.org/10.1371/journal.pntd.0010905 |
| spellingShingle | Manutsanun Inthawong Nattapon Pinthong Areerat Thaiprakhong Tri Wangrangsimakul Piyanate Sunyakumthorn Jennifer Hill Piengchan Sonthayanon Daniel H Paris Susanna J Dunachie Barbara Kronsteiner A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus. PLoS Neglected Tropical Diseases |
| title | A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus. |
| title_full | A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus. |
| title_fullStr | A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus. |
| title_full_unstemmed | A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus. |
| title_short | A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus. |
| title_sort | whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of cd4 t cells in acute scrub typhus |
| url | https://doi.org/10.1371/journal.pntd.0010905 |
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