Tregs attenuate TNF-α production by T lymphocytes within MCMV-infected brains.

For intracellular staining of TNF-α, brain leukocytes (2 x 106 cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with an 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 staining of TNF-α using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (A) Representative plots show the ratio of CD8+ T lymphocytes producing TNF-α 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 TNF-α at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 14 dpi and 30 dpi.