Fig. 5.

MPV immune evasion requires active viral replication. (A and B) To determine whether early gene expression was required for MPV immune suppression of TcR-mediated cytokine responses, PBMCs were cultured in medium or infected with VV or MPV for 12 h in the presence or absence of Arabinoside C (Ara-C) to prevent late gene expression. Anti-CD3 and Brefeldin A were added for an additional 6 h and INγ⁺TNFα⁺ responses in CD4⁺ T cells (A) or CD8⁺ T cells (B) were determined by ICCS and normalized to the values obtained after anti-CD3 stimulation of uninfected cultures. (C) To determine whether nonreplicating MPV was capable of suppressing T cell responses, PBMCs from a representative MPV-immune subject (4 months postinfection) were cultured in Medium or with VV, MPV, UV-inactivated VV (UV-VV), or UV-inactivated MPV (UV-MPV) at a MOI of 0.3. After 12 h stimulation followed by an additional 6 h incubation in the presence of Brefeldin A, CD4⁺ and CD8⁺ T cell responses were determined by ICCS. Dotplots were pregated on CD4⁺ or CD8⁺ T cells and the numbers in the upper right quadrants depict the frequency of virus-specific IFNγ⁺TNFα⁺ T cells per million T cells after background subtraction. Statistical significance in A and B was determined using a two-tailed paired Student's t test.