In order to find out whether Tregs have an influence on the persistent CNS infection we first expanded and functionally activated Tregs using the superagonistic anti-CD28 monoclonal antibody clone D665 (mAb D665) [36] . This mAb, while causing a transient expansion of the total lymph node and splenic T cell population, predominantly expands functional CD4 + Tregs without causing systemic cytokine release [37] . In order to test the efficiency of mAb D665 in young mice, we measured Tregs in a preliminary experiment in uninfected mice 3 days after a single i.p. injection of 100 mg D665 by flow cytometry. The proportion of CD4 + Foxp3 + Tregs increased in the spleen and lymph nodes approximately 2fold ( Fig. 2 A; n = 4; P,0.01). This result indicated that mAb D665 can be used for expansion of Tregs also in young mice.