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

In vitro suppression assay. CD4 + T cells were enriched by negative magnetic selection from rJ2.2-or rJ2.2.M Y135Q -infected brains (pooled from 10 mice) or naive spleens of Foxp3 gfp mice using a CD4 + T cell isolation kit and AutoMACS (both from Miltenyi Biotec) or a mouse CD4 + T cell enrichment kit and Purple EasySep Magnet (both from STEMCELL Technologies). After enrichment of CD4 + T cells, CD4 + GFP  responder and CD4 + GFP + regulatory T cells were then sorted using a FACSDiva or FACSAria cell sorter (BD). For the suppression assay, 3 × 10 4 CFSE-labeled (2.5 µM; Invitrogen) responder T cells from rJ2.2-infected brains or naive spleens were co-cultured with the indicated number of T reg cells and 10 5 irradiated CHB3 cells (2,500 rad) per well, in the presence of 5 µM M133 peptide or 1 µg/ml anti-CD3 mAb in a 96-well round bottom plate. After 66 h, cells were harvested and stained with anti-CD4-PE and anti-Foxp3-Alexa Fluor 647. CD4 + Foxp3  responder cells were analyzed for CFSE dilution by flow cytometry. The division index (DI) was obtained using FlowJo software (Tree Star). The percentage of suppression by T reg cells was calculated as follows: % suppression = 100% × [1  (DI of responders plus T reg cells/DI of responders only)].