Fig. 3.

MPV inhibits T cell responses to VV and Epstein–Barr virus (EBV) in trans. (A) PBMCs from a representative VV-immune subject (16 months after infection) were infected with VV (MOI of 0.3), MPV (MOI of 0.3), or a mixture of VV and MPV at the indicated ratios with VV maintained at an MOI of 0.3 in each case. OPV-specific CD4⁺ T cell responses (Upper) and CD8⁺ T cell responses (Lower) were determined by ICCS after 18 h of stimulation with Brefeldin A added for the last 6 h of stimulation. The numbers in the upper right quadrants represent the frequency of virus-specific IFNγ⁺TNFα⁺ T cells per million T cells identified after background subtraction from control wells containing Medium alone. (B) PBMCs from 4 VV-immune subjects (1 month postinfection) were stimulated with VV and/or MPV as in A. The antiviral T cell response was determined by ICCS and normalized to 100% based on the response to VV alone. The data depict the average ± SD. Statistical significance was determined using a two-tailed paired Student's t test. (C) To determine whether the inhibitory effect of MPV occurred in cis or in trans, EBV-transformed LCLs (EBV) from 3 EBV-seropositive VV-immune subjects were infected for 15 h with VV (VV+EBV) or MPV (MPV+EBV), washed extensively to remove secreted proteins, and then added separately to autologous PBMCs or mixed at a 1:1 ratio (VV+MPV+EBV) before mixing with autologous PBMCs for 6 h in the presence of Brefeldin A to stimulate EBV-specific and OPV-specific T cell responses.