Tregs limit IFN-γ production by T lymphocytes within MCMV-infected brains.

Single cell suspension of brain tissue obtained from MCMV-infected DTx-treated and untreated mice (2–4 animals per time point) were banded on a 70% percoll cushion. Brain leukocytes at the 30–70% percoll interface were collected. For intracellular IFN-γ staining, brain leukocytes (2 x 106 cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with a MCMV-specific, MHC class 1-restricted M45 peptide (6 h at 37°C) and treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD8, and for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (A) Representative plots shows the ratio of CD8+ T lymphocytes producing IFN-γ among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14, & 30 dpi in response to peptide treatment. (B) Pooled data show the (mean ±SD) of CD8+ T-cells producing IFN-γ at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 7, 14, & 30 dpi.