Isolation of CMV-specific CD4⁺ T cells found early and late during primary CMV infection. (A) Top panels show patient 1; bottom panels, patient 2. In the left-hand panels, kinetics of CMV viral load as measured by quantitative PCR (right y-axis) are plotted together with the percentage of CMV-specific IFNγ-producing CD4⁺ T cells (left y-axis). In the right-hand panels, the CMV viral load is plotted together with the percentage of CD4⁺CD28^– T cells (left y-axis). On the x-axes, the time is indicated as weeks after transplantation, which coincides with the moment of infection. The arrows indicate the time points at which CMV-specific IFNγ-producing or CD28^–CD4⁺ T cells were isolated. (B) Sorting gates of CMV-specific CD4⁺ T cells in samples at a late time point after infection. Top panels show gating on lymphocytes and subsequently on CD4⁺CD28^– T cells. Bottom panels show gating on (activated) lymphocytes followed by gating IFNγ-producing CD4⁺ T cells upon stimulation with CMV Ag. Unstimulated cells and SEB-stimulated cells were used to set the gates. Black squares indicate the populations of CMV-specific cells that were isolated. (C) Example of purity of sorted IFNγ⁺ CD4⁺ T cells. Left panel shows sample before sort; right panel shows the result after sorting. Numbers indicate the percentages within the corresponding quadrants.