Cytokine production and proliferation of CD27-sorted T-helper cell subsets. FACS-sorted CD4+CD8α-CD27+ (naïve), CD4+CD8α+CD27+ (CD27+) and CD4+CD8α+CD27- (CD27-) cells were cultured in the presence of 10% CD172a+ APCs. Microcultures were cultivated in Medium alone or stimulated with ConA and rhIL-2 for 4 days. (A) Supernatants of total PBMCs, naïve, CD27+ and CD27--sorted T helper cells were tested for IFN-γ, TNF-α and IL-2 levels by ELISAs. The bar graphs represent mean values + standard deviations of duplicate wells in ng/mL. Representative data of supernatants from one animal out of six is shown. (B) Cytokine production of naïve-, CD27+- and CD27--sorted T helper cells. Cytokine production in supernatants of cell cultures from six pigs is displayed in pg/mL on a logarithmic scale. The coloured bars indicate the mean values of the six individuals for each T-helper cell subset. Significant differences are denoted (* p < 0.05, ** p< 0.01). (C) Total PBMCs, naïve-, CD27+- and CD27--sorted T helper cells were analysed for tritium incorporation at day 3 to 4 of cultivation. The bar graph shows counts per minute (cpm) on the y-axis, representing the mean + standard deviation of triplicate cultures. Data is representative for one experiment with cells from one animal out of two. (D) Naïve- (left column), CD27+- (middle column) and CD27--sorted (right column) T helper cells were labelled with violet proliferation dye prior to cultivation and analysed for proliferation by FCM after four days. Percentage divided and proliferation index (PI) of proliferative cells were determined using FlowJo software and are depicted in the graphs. The dotted lines indicate cells that have not divided as calculated by FlowJo software. Per sample at least 2 × 104 cells were recorded. Data of three animals (top to bottom) are shown.