Proliferative ability of NK-cell subsets in vitro and in vivo after YFVvaccination. (A-B) Analysis of in vitro proliferation of natural killer (NK)–cell subsets (CD56^bright, CD56^dim CD62L⁺, and CD56^dim CD62L⁻ cells) was measured by CFSE dilution after 5 days of stimulation with either 50 ng/mL interleukin-2 (IL-2), IL-15, or IL-12, with myeloid dendritic cells (mDCs) in the presence of 100 ng/mL lipopolysaccharide and 10 μg/mL R848 or with plasmacytoid DC plus 10 μg/mL R848, 10 μg/mL CpG-A, and 10 ng/mL IL-3. (A) Representative data of 10 independent experiments is shown. Stimulated (open histograms) and unstimulated (gray-filled histograms) cells as well as the percentage of proliferating cells are depicted for each condition. (B) Mean percentage ± SEM of proliferating cells after high dose of IL-2 stimulation of 10 independent experiments is shown. P was calculated by Wilcoxon test. (C-D) In vivo proliferation of NK-cell subsets was measured by Ki67 staining of peripheral blood mononuclear cells (PBMCs) derived from healthy donors. (C) One representative donor (top panel) and percentage of Ki67⁺ cells plus corresponding medians of 22 donors analyzed ex vivo (bottom panel) are shown. (D) Ki67 expression was analyzed directly before (day 0), or after yellow fever virus (YFV) vaccination (days 7 and 28). Analysis was performed after gating on total CD3⁻ CD56⁺ NK cells (left and middle) or on CD56^bright (●), CD56^dim CD62L⁺ () and CD56^dim CD62L⁻ (, right). Percentage of Ki67⁺ cells of 8 donors and corresponding median values (left and right graphs) or mean percentage ± SEM of Ki67⁺ cells of 3 donors (middle) are depicted. *P < .05; **P < .01; ***P < .0001 as calculated by Wilcoxon test.