Proliferation and Foxp3 expression in sorted CD4
T cells co-cultivated with PRRSV-infected moDCs.
(A) Temporal progression of experimental procedure: moDCs were harvested 7 days after the start of cultivation and infected with PRRSV-2, PRRSV-1 or mock-infected. Approximately 7 h after infection, violet-stained MACS-sorted CD4+ T cells were added and co-cultivated with moDCs for 4 days. One day after infection, the frequency of N-protein+ moDCs present in the co-cultures was analyzed. After 4 days of co-cultivation, CD4+ T cells were analyzed for proliferation and Foxp3 expression. (B) Gating strategy: doublets and dead cells were excluded by consecutive gating. Violethigh and violetdim cells (representing non-proliferating and proliferating CD4+ T cells, respectively) were gated and analyzed for Foxp3 expression. Foxp3-defined subsets were gated and analyzed for proliferation. (C) Histograms show violet dye fluorescence intensity for total CD4+ T cells (left panel), Foxp3−CD4+ T cells (middle panel) and Foxp3+CD4+ T cells (right panel) in mock-infected (top), PRRSV-2 infected (middle) and PRRSV-1 infected co-cultures (bottom). Bars in histograms represent the border of proliferating and non-proliferating cells. Bold numbers in histograms indicate the percentage of proliferating cells within the respective subset; numbers in brackets show the percentage of proliferating cells within total CD4+ T cells. Exemplary data of CD4+ T cells derived from a single animal is shown. (D) Frequency of Foxp3+ cells within total CD4+ T cells (y-axis) for mock-infected (control co-cult), PRRSV-2 infected (PRRSV-2 co-cult) and PRRSV-1 infected co-cultures (PRRSV-1 co-cult). (E) Frequency of total proliferating cells (left), proliferating Foxp3− (middle) and proliferating Foxp3+ cells (right) within CD4+ T cells (y-axis) for mock-infected co-cultures, PRRSV-2 infected co-cultures and PRRSV-1 infected co-cultures (x-axis). (D + E) Each symbol represents data of CD4+ T cells derived from one individual animal (PRRSV-2: n = 5; PRRSV-1: n = 3). Black bars show the respective mean values.