IFN-?– and IL-10–expressing virus epitope-specific Foxp3(+) T reg cells in the central nervous system during encephalomyelitis

Zhao, Jingxian; Zhao, Jincun; Fett, Craig; Trandem, Kathryn; Fleming, Erica; Perlman, Stanley

Do virus-specific T reg cells generally play a critical role in diminishing immunopathology? In several infectious diseases, including patients infected with West Nile virus or Dengue fever virus, higher T reg cell frequencies correlated with improved outcomes (Lühn et al., 2007; Lanteri et al., 2009) . Relevant for our results, Mtb-specific TCR Tg T reg cells were more potently suppressive than Mtb-nonspecific TCR Tg T reg cells in infected mice (Shafiani et al., 2010) . Furthermore, although transfer of natural T reg cells diminished disease in the setting of autoimmune disease, suppression was estimated to be 20-50-fold more potent if T reg cells were specific for an epitope at a site of inflammation (Tang et al., 2004; Tarbell et al., 2004) . Thus, islet-specific T reg cells prevented diabetes in NOD mice, whereas nonspecific T reg cells at the same dosage could not. If true for rJ2.2-specific T reg cells, these cells may be especially potent in diminishing immunopathology in mice with encephalomyelitis and would be most activated and, thereby, suppressive during acute infection when viral antigen is maximal (Bergmann et al., 2006) . Intracellular cytokine staining. To detect cytokine production by antigen-specific CD4 + effector T cells (T eff cells) and T reg cells, mononuclear cells isolated from the brain and spinal cord were stimulated with 5 µM of peptides M133, S358, M133 + S358 (rJ2.2 infection), or MOG 35-55 (in EAE) in complete RPMI 1640 medium for 6 h at 37°C, in the presence of 1 µl/ml Golgi plug (BD) and antigen-presenting cells (CHB3 cells, B cell line, H-2D b , I-A b ). The rJ2.2-specific epitopes encompass residues 133-147 of the transmembrane (M) protein and residues 358-372 of the surface (S) glycoprotein (Xue and Perlman, 1997; Haring et al., 2001) . Intracellular expression of IFN-, IL-10, TNF, IL-2, or IL-17 was assayed. For some samples, cells were pooled from two to four mice. In some experiments, infected mice were treated with 0.25 mg BFA (Sigma-Aldrich) or vehicle (5% DMSO) intravenously. 6 h later, CNS-derived lymphocytes were analyzed directly ex vivo for IFN- expression as described previously (Liu and Whitton, 2005) . Only infected mice were analyzed because virtually no T cells are detected in the uninfected brain.