Phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice

Regulatory T (T_reg) cells, which express Foxp3 as a transcription factor, are subsets of CD4⁺ T cells. T_reg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of T_regcells is to suppress the proliferation of effector T (T_eff) cells and the production of cytokines such as IFN-γ, TNF-α, and IL-2. It has been demonstrated that T_reg cells' ability to inhibit the function of T_effcells is enhanced during persistent pathogen infection and cancer development. To clarify the function of T_reg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human T_reg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated T_reg cells. To isolate activated T_reg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. T_reg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting T_reg cells isolated from naïve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated T_regcells from resting T_reg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated T_reg cells.