Viral Infection of Transgenic Mice Expressing a Viral Protein in Oligodendrocytes Leads to Chronic Central Nervous System Autoimmune Disease

Evans, Claire F.; Horwitz, Marc S.; Hobbs, Monte V.; Oldstone, Michael B.A.

Chromium Release CTL Assays. 51 Cr-release CTL assays were performed as described (25) . In brief, primary LCMV-specific CTL were generated by i.p. inoculation of 5 ϫ 10 5 PFU of LCMV. Secondary viral CTL assays were done by infecting LCMV-immune mice with viruses as indicated. 7 d later the animals were killed and single cell suspensions were prepared from each of the spleens. Target cells were BALB clone 7 (H-2 d ) and MC57 (H-2 b ) fibroblasts cell lines that were either uninfected or infected 48 h before the assay with LCMV or 12 h before with VV recombinants. The effector cells were plated with the target cells at E/T ratios of 50:1 and 25:1 and incubated at 37 Њ C for 5 h. Effectors were always tested on both MHC-matched and MHCmismatched targets. The supernatants were harvested, and released 51 Cr was measured in a ␥ -counter. The specific 51 Cr release was calculated by the following formula: ([experimental release Ϫ spontaneous release] / [maximum release Ϫ spontaneous release]) ϫ 100. Each variable was tested in triplicate wells, and the variance among triplicates was within 10%.