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Published on: December 13, 2012
Simultaneous cytometric analysis of (auto)antigen-reactive T and B cell proliferation
Sandra Schneider1, Anne Bruns, Beate Moewes
1Universitätsklinikum Charité, Med. Klinik m. S. Rheumatologie und Klinische Immunologie, Berlin, Germany.
Insights
This study optimized a CFSE-based cytometry method to detect antigen-specific B and T cells, crucial for understanding immune responses and diseases.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- Characterizing antigen-specific lymphocytes is vital for studying immunopathologies.
- Current methods require optimization for detecting low-frequency cells.
Purpose of the Study:
- To develop and validate a Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cytometry assay for analyzing antigen-specific lymphocyte proliferation.
- To characterize antigen-reactive B and T cells in response to various stimuli.
Main Methods:
- Peripheral blood mononuclear cells (PBMC) were labeled with CFSE and stimulated with superantigens, recall antigens, allergens, or autoantigens.
- Proliferation was assessed by CFSE dilution via flow cytometry.
- Antigen-specific B cells were phenotyped, and T helper cells were functionally characterized by cytokine secretion.
Main Results:
- The optimized CFSE assay successfully detected antigen-specific proliferated B and T cells, even at low frequencies.
- Distinct cytokine profiles (IL-13, IL-4) were observed in allergen-reactive T cells from atopic vs. healthy donors.
- TT-vaccinated donors showed high frequencies of TT-specific B cells with a plasmablast phenotype.
Conclusions:
- CFSE-based cytometry provides a robust method for detecting and characterizing antigen-specific B and T cells.
- This technique aids in understanding immune responses to various antigens and in identifying disease-related cellular changes.
Abstract:
The detection and characterization of (auto)antigen-specific lymphocytes, both B and T cells, is essential to investigate immunopathologic mechanisms. Our aim was to perform a CFSE (Carboxyfluorescein diacetate succinimidyl ester)-based cytometric analysis of peripheral blood mononuclear cells (PBMC) proliferating in response to antigenic provocation. CFSE-labeled PBMC were stimulated with a superantigen (SEB), a recall antigen (tetanus toxoid), an allergen (grass pollen) and an autoantigen (nucleosomes) and stained after cultivation with CD4-, CD8- and CD19-antibodies. Proliferated cells were identified cytometrically by the decrease of the CFSE fluorescence intensity due to cell division. Antigen-reactive, proliferated B cells were further analysed phenotypically, antigen-specific proliferated Th cells were further characterized functionally regarding their cytokine secretion pattern after polyclonal restimulation. Using this technique, antigen-specific proliferated B and Th cells were detected even at low frequencies. Analyzing the cytokine secretion pattern of allergen-reactive proliferated Th cells after polyclonal restimulation we found differences in the expression of IL-13 and IL-4 between an atopic and a healthy donor. After stimulation of PBMC from TT-vaccinated donors TT-specific proliferated B cells were detected in high frequencies and showed a plasmablast-typical CD20(low) CD27(high) phenotype with only low frequencies expressing CD138 (= Syndecan-1). Proliferation of nucleosome-reactive Th cells and B cells was observed in both patients and healthy controls. We have optimized here the cytometric analysis of reactive cell proliferation based on CFSE offering various facilities of application on the further characterization of both antigen-specific B and T cells.
