Attenuation of GARP expression on regulatory T cells by protein transport inhibitors

Abstract An integrated understanding of the functional capacities of cells in the context of their physical parameters and molecular markers is increasingly demanded in immunologic studies. Regulatory T cells (Tregs) are a subpopulation of T cells involved in immune response modulation and mediating tolerance to self-antigen with their absence leading to a loss of tolerance. Glycoprotein repetitions A predominant (GARP) is a key marker for activated Tregs, but its detection may also be useful in determining the functional capacities of the cell. This study aims to deduce the optimal stimulation period and the impact of protein transport inhibitors (PTIs), commonly used in the detection of intracellular cytokines, on GARP detection. Through flow cytometric analysis we analyzed different cell culture conditions for optimal GARP expression on activated Tregs. Healthy donor PBMCs were stimulated with either Staphylococcal Enterotoxin B (SEB) or PMA/Ionomycin (PMA/Iono), in the presence and absence of PTIs monensin and/or brefeldin A (BFA) and GARP expression was assessed on CD4+ CD25+ FOXP3+ Tregs. The optimal stimulation period for the detection of GARP was highest at 24-h. Furthermore, we determined that GARP expression on Tregs is significantly reduced when cells are treated with the PTIs monensin and/or BFA following PMA/Iono stimulation. This effect was not seen following SEB stimulation. Therefore, due to the effects of PTIs, alternative methods should be considered when performing simultaneous analysis for cytokine expression and GARP expression on Tregs.