Regulatory T cells represent a FOXP3+CD25+ subset of CD4+ T cells that play a crucial role in the establishment and maintenance of immune tolerance. Owing to their immunosuppresive function, they are also critically involved in the immunopathogenesis of autoimmune diseases, transplant rejection and graft-versus-host disease. The development of therapies for those conditions is increasingly focused on Tregs, which represent a promising therapeutic approach, as currently available treatments are largely based on non-specific immunosuppression and are associated with numerous adverse effects. A major challenge in the development of Treg based therapies remains the lack of specific surface markers that would enable improved characterization of these cells. The aim of this thesis was to evaluate the expression of surface markers on Treg cells based on previous RNA sequencing results, identify potential specific surface markers, and determine the temporal dynamics of their expression during ex vivo expansion. The experimental work included the isolation of Tregs from mouse spleens using immunomagnetic separation, their activation with anti-CD2/CD28 activation beads, and ex vivo expansion for up to fifteen days. To achieve a more comprehensive characterization of Tregs, the existing antibody panel was expanded with antibodies against new markers that further define the phenotype of ex vivo expanded Tregs. Tregs were stained with fluorescently labeled antibodies and viability dye and analyzed by spectral flow cytometry. The analysis focused on four new markers that showed co-expression with FOXP3, the main transcription factor that defines the identity of Treg cells. A gradual decline in FOXP3 expression was observed over time, suggesting a progressive loss of Treg and the expansion of conventional CD4+ T cells. Comparison of cytograms demonstrated that the expression of the investigated markers on Tregs was most expressed on day 4, corresponding to the activation phase of Tregs. Among the analyzed markers, marker 1 exhibited the highest level of expression on Tregs and low expression on the remaining CD4+ T cell population, indicating good specificity for the Treg population. These results provide a foundation for further investigation of the biological significance of marker 1 and its potential application in the improved identification and characterization of Tregs.
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