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

TCR Rg mice were engineered as previously described (Holst et al., 2006 ). An M133-specific TCR- (V8.3, TRBV13-1) identified in three of three mice was produced synthetically (Geneart) and then cloned into a retroviral vector containing an IRES-GFP cassette to allow identification of transfected cells by GFP expression. Retroviral producer cell lines expressing this TCR- were produced and used to transduce B6 donor bone marrow before reconstitution of lethally irradiated B6 recipient mice. After reconstitution, these Rg mice were infected with rJ2.2. The majority of CD4 T cells in the infected CNS were GFP + . I-A b /M133 tetramer + GFP + cells were sorted from the infected CNS and analyzed for TCR- chain expression. Two TCR- chains (V2.3 and TRAV14-1), differing by a single amino acid in the CDR3 region, were detected using RT-PCR-based cloning. One of these TCR- was synthesized and cloned into the same retroviral vector described in this section. In this vector, the TCR- and TCR- chains are linked via the 2A protease, facilitating cleavage. Because the two chains are linked on the same molecule, they are produced in stoichiometric amounts. TCR Rg mice expressing both M133-specific TCR- and TCR- were produced. Epitope M133 specificity was confirmed by analysis of GFP + cells in the infected CNS using M133-specific tetramer and IFN- expression after peptide stimulation.