Looking for Tregs in the brain, we did not detect a significant number of CD4 + Foxp3 + Tregs by FACS-analysis after staining with Foxp3-specific antibodies, and only a small number of GFP- Figure 3 . Expansion of T lymphocytes with the superagonistic CD28 antibody D665 induces virus replication and spread. Consecutive coronal brain sections (100 mm sections) were prepared from complete rMV-green-infected mouse cerebra and analyzed using the UV microscope. Overviews and details of a typical section of an infected brain of a mouse treated with mAb D665 and analyzed at 28 dpi (A), and sections of infected control animals in the absence of mAb D665 at 28 dpi (B), and 14 dpi (C) are shown. The numbers of infected eGFP + cells per brain (sections through the complete cerebrum of each animal were evaluated as described [33] ) were determined microscopically in infected control C57BL/6 mice at 7, 14, 28 and 42 dpi (D, lanes 1-4) and in D665-treated mice at 28 and 42 dpi (D, lanes 5 and 6). The difference between control and D665-treated mice at 28 dpi was highly significant (P,0,0001). doi:10.1371/journal.pone.0033989.g003 expressing Foxp3 + cells in infected DEREG mice. These findings suggest that only very few Tregs, if any at all, are required within the brain for the regulation of effector immune cells, and that this regulation predominantly takes place in secondary lymphoid organs. Sellin et al. observed an increase of Foxp3 + Tregs as a consequence of MV infection of CD150-transgenic mice in spleen and brain [32] , which suggests that also the MV-infection itself supports the expansion and migration of Tregs, and that these infection-induced Tregs may be part of the multifactorial MVinduced immunosuppression [3, 4] . Tregs can restrain effector T cell responses through the production of immunomodulatory cytokines, such as TGF-b, IL-10, and IL-35, expression of inhibitory ligands, such as CTLA-4 and LAG-3, cytokine consumption, and direct cytolysis. It remains to be elucidated which of these mechanisms are involved in the persistent brain infection with MV.