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Updated: Jul 12, 2025
High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues
Published on: September 15, 2021
Combinatorial multimer staining and spectral flow cytometry facilitate quantification and characterization of
Dennis Hoving1, Alexandre H C Marques2, Wesley Huisman2
1Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. d.hoving@lumc.nl.
Insights
Researchers developed a new method to study B cells that respond to bacterial capsular polysaccharides, crucial for vaccines. This technique reveals how immune cell characteristics vary by vaccine type and geographic origin.
Area of Science:
- Immunology
- Microbiology
- Vaccinology
Background:
- Bacterial capsular polysaccharides are key vaccine targets.
- Studying immune responses to these polysaccharides is challenging due to technical limitations.
Purpose of the Study:
- To develop and validate a high-throughput assay for analyzing antigen-specific B cells.
- To enable in-depth characterization of polysaccharide-specific B cells and their immune responses.
Main Methods:
- Developed a method using combinatorial staining with fluorescently-labeled capsular polysaccharide multimers.
- Simultaneously analyzed 14 Streptococcus pneumoniae and 5 Streptococcus agalactiae serotype-specific B cell populations.
- Utilized concurrent staining of 25 cellular markers for comprehensive cell characterization.
Main Results:
- Phenotype of polysaccharide-specific B cells correlated with serotype specificity, vaccination history, and donor population.
- Observed a link between non-class switched (IgM+) memory B cells and vaccine-inefficient S. pneumoniae serotypes 1 and 3.
- Found increased B cell activation in South African donors compared to Dutch donors, potentially linked to S. agalactiae incidence.
Conclusions:
- The developed assay allows for detailed characterization of B cell heterogeneity.
- This heterogeneity may explain variations in vaccine efficacy.
- Provides insights into immune responses against encapsulated bacterial pathogens.
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.
