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Combinatorial multimer staining and spectral flow cytometry facilitate quantification and characterization of polysaccharide-specific B cell immunity.

ABSTRACT: Bacterial capsular polysaccharides are important vaccine immunogens. However, the study of polysaccharide-specific immune responses has been hindered by technical restrictions. Here, we developed and validated a high-throughput method to analyse antigen-specific B cells using combinatorial staining with fluorescently-labelled capsular polysaccharide multimers. Concurrent staining of 25 cellular markers further enables the in-depth characterization of polysaccharide-specific cells. We used this assay to simultaneously analyse 14 Streptococcus pneumoniae or 5 Streptococcus agalactiae serotype-specific B cell populations. The phenotype of polysaccharide-specific B cells was associated with serotype specificity, vaccination history and donor population. For example, we observed a link between non-class switched (IgM⁺) memory B cells and vaccine-inefficient S. pneumoniae serotypes 1 and 3. Moreover, B cells had increased activation in donors from South Africa, which has high-incidence of S. agalactiae invasive disease, compared to Dutch donors. This assay allows for the characterization of heterogeneity in B cell immunity that may underlie immunization efficacy.

SUBMITTER: Hoving D

PROVIDER: S-EPMC10613281 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Combinatorial multimer staining and spectral flow cytometry facilitate quantification and characterization of polysaccharide-specific B cell immunity.

Hoving Dennis D Marques Alexandre H C AHC Huisman Wesley W Nosoh Beckley A BA de Kroon Alicia C AC van Hengel Oscar R J ORJ Wu Bing-Ru BR Steenbergen Rosanne A M RAM van Helden Pauline M PM Urban Britta C BC Dhar Nisha N Ferreira Daniela M DM Kwatra Gaurav G Hokke Cornelis H CH Jochems Simon P SP

Communications biology 20231028 1

Bacterial capsular polysaccharides are important vaccine immunogens. However, the study of polysaccharide-specific immune responses has been hindered by technical restrictions. Here, we developed and validated a high-throughput method to analyse antigen-specific B cells using combinatorial staining with fluorescently-labelled capsular polysaccharide multimers. Concurrent staining of 25 cellular markers further enables the in-depth characterization of polysaccharide-specific cells. We used this a ...[more]

PMID: 37898698

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Project description:BackgroundQualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity.MethodsWhole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period.ResultsThe limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells.ConclusionsThe whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Dataset Information

Combinatorial multimer staining and spectral flow cytometry facilitate quantification and characterization of polysaccharide-specific B cell immunity.

Publications

Combinatorial multimer staining and spectral flow cytometry facilitate quantification and characterization of polysaccharide-specific B cell immunity.

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