Detection of pp65 specific CD4+ and CD8+ T cell responses.

<p>PBMC from 100 healthy donors, 44 CMV seronegative and 56 seropositive, were analyzed either <i>ex vivo</i> or after <i>in vitro</i> stimulation with the pp65 peptide mixture for 7 days. The cells were examined for pp65 specific responses using the ICS assay and stained for CD4, CD8, CD69 and intracellular TNFα and IFNγ collectively. Background activation obtained in the absence of peptide was subtracted from the results obtained with the peptide mixture. In A) is shown examples of the ICS analysis of CD4 and CD8 T cell responses measures <i>ex vivo</i> and after stimulation of both a seropositive and a seronegative donor. In B) the <i>ex vivo</i> measured frequency of pp65 specific CD4 and CD8 T cells is shown for each seronegative and seropositive donor, respectively. In C) the frequency of pp65 specific CD4+ and CD8+ T cells measured after <i>in vitro</i> stimulation is shown for each seronegative and seropositive donor, respectively. After <i>in vitro</i> stimulation, the background activation was slightly higher than observed <i>ex vivo</i>; positive responses were defined as frequencies >0.09% for the <i>ex vivo</i> analysis, and as frequencies >0.3% for the <i>in vitro</i> stimulation analysis. Background frequencies have been subtracted the specific frequencies and responses above 0.09% for the ex vivo analysis and 0.3% for the in vitro analysis were considered positive. Shown as the responses above the grey areas.