Using flow cytometric analysis of a previously characterized preparation of brain-infiltrating leukocytes (BILs) (Howe et al, 2007) , we measured the number of NKG2D-positive immune cells present in the CNS at 3 and 7 days after TMEV infection (2×10 5 PFU i.c.; 4-6 week old C57Bl/6J mice; all experiments adhered to Mayo IACUC guidelines). We found that at 3 days postinfection (dpi) 6 ± 0.4% of CD45 hi BILs were CD8-positive, of which 20 ± 0.6% were also positive for the NKG2D costimulatory receptor. By 7 dpi the BILs were comprised of 24 ± 2% CTLs, with 76 ± 2% of these cells positive for NKG2D (n=4-6 mice per timepoint; mean ± 95% CI). Thus, between 3 and 7 dpi the phenotype of immune cells infiltrating the CNS shifted toward a population that was enriched in NKG2D + CD8 + CD45 hi CTLs ( Figure 1A -1F). The presence of NKG2D-positive CTLs within the infected hippocampus (Wada and Fujinami, 1993) was confirmed by simultaneously immunostaining cryosections from mice at 7 dpi with anti-CD8 (53-6.7, 1:100) and anti-NKG2D (CX5, 1:50) (Figure 2A -2E) (Howe et al, 2004) .