Supplementary Material for: Immune Dysregulation in Chronic Stress: A Quantitative and Functional Assessment of Regulatory T Cells

Objective: Chronic stress is closely related to immune dysfunction. Immune parameters have been analyzed in many ways in humans and animals under chronic stress. Recently, it has been proved that FoxP3+ regulatory T cells (Tregs) play a key role in immune regulation in vivo. However, it has not yet been elucidated how Tregs respond to chronic stress in vivo. Therefore, in the present study, we investigated the frequency of and functional changes in Tregs from mice under chronic stress. Methods: Spleen cells were separated from C57/BL6 mice that had been exposed to immobilization stress for 3 weeks. The frequencies of FoxP3+ and CD4+ CD25+ cells were analyzed by flow cytometry. CD4+CD25– cells (effector T cells, Teffs), CD4+CD25+ cells (Tregs) and CD4– cells (antigen-presenting cells, APCs) were separated for the functional assessment of the proliferative activity of Teffs, the suppressive activity of Tregs and the feeder activity of APCs. Results: The results showed that chronic immobilization stress significantly increased the frequencies of CD4+CD25+ and CD4+FoxP3+ cells. Chronic immobilization stress also enhanced the suppressive function of CD4+ CD25+ Tregs. On the other hand, the proliferative activity of Teffs and the feeder activity of APCs were decreased in the mice under chronic immobilization stress. Conclusion: Taken together, it is suggested that increased number and function of Tregs may actively contribute to the immune dysfunction in chronic immobilization stress, synergizing with the decreased function of Teffs and APCs.