Regulatory T cells are a subset of CD4+ T cells characterized by the expression of the transcription factor FOXP3 and high expression of CD25. They play a crucial role in maintaining immune homeostasis by suppressing the activity of conventional effector T cells, thereby preventing excessive immune responses and the development of autoimmunity. The aim of this master's thesis was to perform an in vitro suppression assay and establish a multiparametric system based on spectral flow cytometry for the investigation of the immunosuppressive capacities of regulatory T cells. Regulatory and conventional T cells were isolated from murine spleens using an optimized isolation protocol. The cells were activated either with activation beads or with a cell stimulation cocktail, while the control group remained unstimulated. The activation and proliferation of conventional T cells were compared in the absence and presence of regulatory T cells. By staining cellular markers indicative of cell activation and using the CFSE dye to monitor proliferation, the immunosuppressive capacity of regulatory T cells in co-culture was evaluated using spectral flow cytometry. The presence of regulatory T cells in samples activated with beads significantly reduced the expression of the activation markers CD25 and CD69 and affected proliferation modeling parameters, including the number of undivided cells and the replication index in bead-activated samples, as well as the percentage of divided cells in samples activated with the cell stimulation cocktail. The in vitro immunosuppression assay represents the first step in studying the suppressive capacity of regulatory T cells. The knowledge gained from this assay serves as a foundation for subsequent in vivo studies while also enabling the transfer of the methodology to other cellular systems, such as human or genetically modified cells.
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