Simultaneous cytometric analysis of (auto)antigen-reactive T and B cell proliferation.

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.