Figure 5.

T reg cells of infants have greater suppressive activity than those of adults. (A) Representative flow cytograms showing intracellular FOXP3 staining in uninfected adult (left) and infant (right) peripheral blood cells. Numbers represent the percentage of gated events found in each quadrant. (B) Percentages of CD3⁺CD4⁺CD25⁺ cells from uninfected peripheral blood that were shown to express FOXP3 by intracellular staining and flow cytometry. Horizontal bars represent means. (C) Suppression of cell division by CD25⁺ T reg cells from infant and adult macaques isolated using paramagnetic beads. Data points indicate the division index achieved by CFSE-labeled cells in each sample as a percentage of the maximal value obtained in the CD25-depleted population. Cells were isolated from peripheral blood, purified using paramagnetic beads and columns, and stimulated for 5 d on anti-CD3–coated tissue culture plates. These data are representative of two experiments that tested a full range of T reg cell/responder ratios from infants and adults in parallel and are consistent with a much larger number of results testing suppression at a ratio of 1:3 and shown in D. (D) Results from a larger number of suppression assays performed on different days and testing suppression at a 1:3 ratio of T reg cells/responders. Horizontal bars represent means. (E) Adult and infant T reg cell populations purified by FACS. T reg cells represented >90% of the CD3⁺CD4⁺ cells in sorted populations. The far right panel shows the intensity of FoxP3 staining in aliquots of sorted adult and infant T reg cells (mean fluorescence intensity = 513 and 831, respectively). (F) Results of suppression assays performed using regulatory cells purified by FACS from the blood of two uninfected infants and two uninfected adults. Because of limited cell numbers, these assays were performed using only 40,000 CD25-depleted responders. Responders were depleted of CD25⁺ cells using paramagnetic beads. (G) Effect of T reg cell depletion on cytokine production by infant T cells in response to polyclonal stimuli. Aliquots of infant T cells were depleted of CD25⁺ cells using paramagnetic beads. Depleted or undepleted samples were then stimulated with PMA and ionomycin for 12 h, permeabilized, and stained with antibodies specific for cytokines or cell-surface markers. The percentage of CD8⁺ (far left graph) or CD4⁺ (right three graphs) expressing various cytokine combinations (simultaneous IFN-γ and TNF-α) or individual cytokines (IFN-γ, IL-2, or IL-4) in response to stimulation is shown. At least 12 samples are shown in each graph.